Tissue plasminogen activator binds to human vascular smooth muscle cells by a novel mechanism. Evidence for a reciprocal linkage between inhibition of catalytic activity and cellular binding.

Human vascular smooth muscle cells (VSMC) bind tissue plasminogen activator (tPA) specifically, saturably, and with relatively high affinity (K(d) 25 nM), and this binding potentiates the activation of cell-associated plasminogen (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322). We have observed that this binding can be efficiently competed by DFP-inactivated tPA and S478A-tPA but not by tPA inactivated with H-D-Phe-Pro-Arg-chloromethyl ketone (PPACK). VSMC-bound tPA also exhibited a markedly reduced inhibition by PPACK, displaying biphasic kinetics with second-order rate constants of 7. 5 x 10(3) M(-1) s(-1) and 0.48 x 10(3) M(-1) s(-1), compared with 7. 2 x 10(3) M(-1) s(-1) in the solution phase. By contrast, tPA binding to fibrin was competed equally well by all forms of tPA, and its inhibition was unaltered. These effects were shown to extend to the physiological tPA inhibitor, plasminogen activator inhibitor 1. tPA.plasminogen activator inhibitor 1 complex did not compete tPA binding to VSMC, and the inhibition of bound tPA was reduced by 30-fold. The behavior of the various forms of tPA bound to VSMC correlated with conformational changes in tPA detected by CD spectroscopy. These data suggest that tPA binds to its specific high affinity site on VSMC by a novel mechanism involving the serine protease domain of tPA and distinct from its binding to fibrin. Furthermore, reciprocally linked conformational changes in tPA appear to have functionally significant effects on both the interaction of tPA with its VSMC binding site and the susceptibility of bound tPA to inhibition.     

Functional characterization of monoclonal antibodies directed against fibrin binding domains of tissue-type plasminogen activator

Fibrin interacts with tissue-type plasminogen activator (tPA) via the finger and the kringle 2 domains. Three monoclonal antibodies against tPA, designated MPW3VPA, MPW6VPA, and MPW7VPA, which react with epitopes in the tPA molecule involved in fibrin binding, were characterized. The IgM monoclonal antibody MPW6VPA, directed against an epitope close to the finger and epidermal growth factor domains, stimulated plasminogen activation only in the absence of CNBr-fibrinogen fragments by increasing kcat in a dose-dependent fashion, an effect which was not restricted to the intact molecule. These results suggest that MPW6VPA mimics the initial effect of fibrin bound to the tPA molecule, which results in a change of kcat values. The MPW6VPA effect was reversed by another antibody, MPW3VPA, also directed against epidermal growth factor and finger domains. The latter antibody also inhibited plasminogen activation by tPA in the presence of CNBr-fibrinogen fragments in a dose-dependent, apparently noncompetitive way. No effect of MPW3VPA was seen in the absence of CNBr-fibrinogen fragments. MPW7VPA directed against kringle 2 of tPA inhibited plasminogen activation by tPA only when CNBr-fibrinogen fragments were present. This inhibition was apparently competitive and dose-dependent. These data suggest that MPW3VPA interferes with the first phase of fibrin binding to tPA, whereas MPW7VPA interferes with the second phase of fibrin binding to the tPA molecule via kringle 2, resulting in Km changes.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: a key event in melanoma cell invasiveness in vitro.

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on MeWo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: A key event in melanoma cell invasiveness in vitro

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on Me Wo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Urokinase-type and tissue-type plasminogen activators are essential for in vitro invasion of human melanoma cells

This study evaluates the contribution of two types of plasminogen activators (PAs; tissue-type PA (tPA) versus urokinase-type PA (uPA) toward the invasiveness of human melanoma cells in a novel in vitro assay. We identified two human melanoma cell lines, MelJuso and MeWo, expressing uPA or tPA as shown at mRNA, protein, and enzyme activity level. MelJuso cells produced uPA as well as plasminogen activator inhibitor-1 (PAI-1). The latter was, however, not sufficient to neutralize the cell-associated or secreted uPA activity. MeWo cells secreted tPA, but the enzyme was not found to be cell-associated. PAI-1 production by these cells was not detectable. Plasminogen activation and fibrinolytic capacity of both cell lines were reduced by anticatalytic monoclonal antibodies specific for the respective type of PA or by aprotinin. In a novel in vitro invasion assay, antibodies to PA as well as aprotinin decreased the invasiveness of both cell lines into a fibrin gel, Matrigel, or intact extracellular matrix. Our results confirm the importance of uPA-catalyzed plasminogen activation in tumor cell invasiveness. Furthermore, we provide evidence that tPA, beyond its key role in thrombolysis, can also be involved in in vitro invasion of human melanoma cells.     

Plasminogen activation is stimulated by prion protein and regulated in a copper-dependent manner

Prion diseases are associated with the conversion of the normal prion protein, PrP(C), to the infectious disease form PrP(Sc). Discrimination between these isoforms would significantly enhance diagnosis of these diseases, and it has recently been reported that PrP(Sc) is specifically recognized by the serine protease zymogen plasminogen (Fischer et al. (2000) Nature 408, 479). Here we have tested the hypothesis that PrP is a regulator of the plasminogen activation system. The effect of recombinant PrP, either containing copper (holo-PrP) or devoid of it (apo-PrP), on plasminogen activation by both uPA and tPA was determined. PrP had no effect on plasminogen activation by uPA. By contrast, the activity of tPA was stimulated by up to 280-fold. This was observed only with the apo-PrP isoforms. The copper-binding octapeptide repeat region of PrP was involved in the effects, as a mutant lacking this region failed to stimulate plasminogen activation, although a synthetic peptide corresponding to this region was unable to stimulate tPA activity. Competition experiments demonstrated that, in addition to plasminogen binding, the stimulation required a high-affinity interaction between tPA and PrP (K(d) < 2.5 nM). Kinetic analysis revealed a template mechanism for the stimulation, suggesting independent binding sites for tPA and plasminogen. Lack of copper-binding may be an early event in the conversion of PrP(C) to PrP(Sc), and our data therefore suggest that tPA-catalyzed plasminogen activation may provide the basis for a sensitive detection system for the early stages of prion diseases and also play a role in the pathogenesis of these diseases.     

Antibody-directed fibrinolysis. An antibody specific for both fibrin and tissue plasminogen activator

An investigation was made to determine whether it is possible to attract tissue plasminogen activator (tPA) to the site of a thrombus by means of an antibody with affinites for both tPA and fibrin. A bispecific antibody conjugate was constructed by cross-linking two monoclonal antibodies: one specific for tPA, the other specific for fibrin. The bispecific antibody enhanced fibrinolysis by capturing tPA at the site of a fibrin deposit. In an in vitro quantitative fibrinolysis assay, the relative fibrinolytic potency of tPA bound to the bispecific antibody was 13 times greater than that of tPA and 200 times greater than that of urokinase. When fibrin was treated with the bispecific antibody before being mixed with tPA, the relative fibrinolytic potency of tPA was enhanced 14-fold. This capture also occurred when the concentration of tPA present in the assay was less than the concentration of tPA present in normal human plasma. In a human plasma clot assay, samples containing both the bispecific antibody and tPA exhibited significantly more lysis than did samples containing tPA alone. In spite of the increased clot lysis effected by the presence of bispecific antibody, there was no significant increase in fibrinogen or alpha 2-antiplasmin degradation at equal tPA concentrations. The ability of the bispecific antibody to concentrate exogenous tPA in vivo was then examined in the rabbit jugular vein model. Systemic infusion of a small amount of tPA (10,000 units) produced no significant increment in thrombolysis over the level of spontaneous lysis (14 +/- 8%). However, the simultaneous infusion of 10,000 units of tPA and 2 mg of bispecific antibody resulted in 42 +/- 14% (p less than 0.01) lysis. These results suggest that a molecule capable of binding both fibrin and tPA with high affinity could enhance thrombolysis in the circulation by capturing endogenous tPA.     

Stimulation of cell surface plasminogen activation by membrane-bound melanotransferrin: a key phenomenon for cell invasion

The activation of plasminogen at the cell surface is a crucial step in cell migration and invasion. In the present study, the effect of membrane-bound melanotransferrin (mMTf), also known as human melanoma antigen p97, on cell surface plasminogen binding and activation was investigated by using Chinese Hamster Ovary (CHO) cells transfected with full-length melanotransferrin (MTf) cDNA and SK-MeL-28 melanoma cells. The expression of mMTf in CHO increased cell surface plasminogen binding by about 2-fold. In addition, application of the monoclonal antibody L235 against MTf as well as truncated, soluble MTf (sMTf) abolished plasminogen binding to MTf-transfected and SK-MeL-28 cells, indicating that mMTf is a potential cell surface plasminogen receptor. Moreover, mMTf expression in CHO cells stimulates plasminogen activation at the cell surface by about 2.5-fold. In addition to the induced binding and activation of plasminogen, cell motility, migration and invasion were about 3-fold higher in CHO cells expressing mMTf. Both monoclonal antibody L235 and truncated sMTf inhibited mMTf-stimulated CHO cell motility, migration and invasion. Overall, our results indicate a key role for mMTf in cell surface plasminogen binding and in activation processes involved during cell migration and invasion.     

Antibody-directed fibrinolysis: a bispecific (Fab')2 that binds to fibrin and tissue plasminogen activator

A bispecific (Fab')2 molecule was constructed by linking the monovalent Fab' from an anti-fibrin monoclonal antibody to the Fab' from an anti tissue plasminogen activator (tPA, single chain) monoclonal antibody by means of inter-heavy-chain disulfide bonds. An immunochemical complex composed of the bispecific (Fab')2 molecule bound to tPA [tPA-bispecific (Fab')2 complex] was then generated and purified. Its molecular weight was 170 kDa [less than half the molecular weight of a previously described tPA-bispecific antibody complex containing the entire anti-fibrin and anti-tPA immunoglobulin molecules; Runge, M. S., et al. (1987) Trans. Assoc. Am. Phys. 100, 250-255]. The tPA-bispecific (Fab')2 complex was 8.6-fold more efficient in fibrinolysis than tPA alone and 94-fold more potent than urokinase. This enhancement in the fibrinolytic potency of tPA compares favorably with that observed for the bispecific whole-antibody complex. These results suggest that this smaller, less immunogenic molecule is capable of binding both fibrin and tPA with high affinity and of enhancing the thrombolytic efficiency of exogenous and, perhaps, endogenous tPA.     

Allosteric regulation of tPA-mediated plasminogen activation by a modifier mechanism: evidence for a binding site for plasminogen on the tPA A-chain.

We studied the mechanism responsible for nonlinear double reciprocal plots for tissue type plasminogen activator (tPA)-mediated plasminogen activation reported previously by several groups. We found nonlinear Eadie-Scatchard plots for Glu-plasminogen activation by recombinant single-chain tPA confirming a non-Michaelis-Menten behavior of tPA. In order to characterize this mechanism, enzyme kinetic studies with truncated substrates (Lys- and miniplasminogen) and modified or truncated enzymes (two-chain tPA and tPA B-chain) were performed. Thereby it could be excluded that product-mediated modifications of the enzyme or the substrate are responsible for the nonlinear plots. Linear plots, i.e., Michaelis-Menten kinetics, were only found when tPA B-chain was used as a plasminogen activator, indicating that the tPA A-chain should be responsible for the non-Michaelis-Menten behavior. Binding studies of plasminogen to immobilized tPA A-chain in fact demonstrated a saturable binding of Glu- and miniplasminogen to the A-chain of tPA with a KD approximately 0.1 microM and one binding site per molecule of tPA A-chain. These data suggested a modifier mechanism responsible for the nonlinear plots whereby the substrate plasminogen itself could function as a modifier. When such a mechanism was included into a model for tPA-mediated plasminogen activation, the experimentally obtained data could be fitted into such a model by nonlinear regression analysis with resulting p-values of less than 0.001.     

Conjugation and evaluation of 7E3 x P4B6, a chemically cross-linked bispecific F(ab')2 antibody which inhibits platelet aggregation and localizes tissue plasminogen activator to the platelet surface.

A bispecific F(ab')2 monoclonal antibody which recognizes both the platelet GPIIb/IIIa receptor and human tissue plasminogen activator was produced to target tPA to platelets for enhancement of thrombolysis. A stable, thioether-cross-linked bispecific F(ab')2 (7E3 X P4B6) combining the GPIIb/IIIa-specific monoclonal antibody 7E3, which inhibits platelet aggregation, and a nonneutralizing anti-tPA monoclonal antibody (P4B6) was produced. This was performed by coupling each of the parental Fab' moieties with the homobifunctional cross-linker bis(maleimido methyl) ether (BMME). 7E3 X P4B6 was sequentially purified using gel-filtration chromatography and hydrophobic interaction (HIC) HPLC. HIC was shown to completely resolve each of the parental F(ab')2 species from the bispecific one. 7E3 X P4B6 was shown to retain completely each of the parental immunoreactivities in GPIIb/IIIa and tPA binding EIA's. The bispecific antibody inhibited platelet aggregation in vitro at levels comparable to those for 7E3 Fab. Recruitment of tPA activity to washed human platelets was demonstrated using the S-2251 chromogenic substrate assay. 7E3 X P4B6 recruited 12-fold more tPA to the washed platelets than a mixture of the parental F(ab')2 molecules used as controls.     

Conjugation to an antifibrin monoclonal antibody enhances the fibrinolytic potency of tissue plasminogen activator in vitro

Tissue plasminogen activator (tPA) was covalently linked by disulfide bonds to a monoclonal antibody specific for the amino terminus of the beta chain of fibrin (antibody 59D8). The activity of the tPA-59D8 conjugate was compared with that of tPA, urokinase (UK), and a UK-59D8 conjugate. For lysis of fibrin monomer, tPA was 10 times as potent as UK, whereas both UK-59D8 and tPA-59D8 conjugates were 100 times as potent as UK and 10 times as potent as tPA. Conjugation of tPA or UK to antibody 59D8 produced a 3.2-4.5-fold enhancement in clot lysis in human plasma over that of the respective unconjugated plasminogen activator. However, the UK-59D8 conjugate was only as potent as tPA alone. Antibody-conjugated tPA or UK consumed less fibrinogen, alpha 2-antiplasmin, and plasminogen than did the unconjugated activators, at equipotent fibrinolytic concentrations. Antibody targeting thus appears to increase the concentration of tPA in the vicinity of a fibrin deposit, which thereby leads to enhanced fibrinolysis.     

Characterization of monoclonal antibodies against human tissue plasminogen activator (tPA): quantitation of free tPA in human cell cultures by an ELISA.

Seven murine monoclonal antibodies produced against tissue plasminogen activator (tPA) were evaluated by means of enzyme-linked immunosorbent assays (ELISAs), and their effects on the enzymatic activities of tPA towards a synthetic substrate (S-2288) and plasminogen were investigated. One of the antibodies, TPA1-70, strongly inhibited the enzymatic activity of tPA in a fibrin agarose plate assay, while it did not affect the enzymatic activity towards the synthetic substrate or plasminogen. The antibody is directed to an epitope on the B-chain of tPA, which is necessary for the formation of a ternary complex of tPA, fibrin and plasminogen, but probably not to the active site. Another antibody, TPA2-14, partially inhibited the enzymatic activities of tPA towards the synthetic substrate and plasminogen, but it was not able to bind to the inactive tPA complexed with plasminogen activator inhibitor-1 (PAI-1). The antibody is directed to an epitope on the second kringle region, which is probably one of the PAI-1 binding sites. This property of the antibody enabled us to develop an ELISA for selective quantitation of free tPA in culture media conditioned with several human cell lines. The results indicate that tPA in these media exists either partially or almost entirely in a complex with PAI-1.     

Protein S attenuates the invasive potential of THP-1 cells by interfering with plasminogen binding on cell surface via a protein C-independent mechanism

Protein S, a cofactor for activated protein C (aPC) to inactivate coagulation factors, also plays a pivotal role in inflammation. Based on our recent findings that aPC and protein S modifies tissue plasminogen activator (tPA)-catalyzed activation of Glu-plasminogen (Glu-plg), we analyzed possible role of protein S in cell-associated plasminogen activation and invasive potential of inflammatory cells. Monocyte-like THP-1 cells, to which both plasminogen and tPA bind, enhanced tPA-catalyzed plasminogen activation, which was partially abolished by protein S but not by aPC. Protein S attenuated both the plasminogen binding to THP-1 cells and associated their invasive potential through Matrigel.     

Release of tissue-type plasminogen activator by activated rat eggs and its possible role in the zona reaction.

The resumption of meiosis results in synthesis of tissue-type plasminogen activator (tPA) in the rat and mouse oocytes (Haurte et al., Cell 43:551-558, 1985). The present study demonstrates that freshly ovulated rat oocytes released their tPA into the surrounding medium upon in vitro activation by sperm penetration or treatment with a calcium ionophore. The presence of a neutralizing monoclonal anti-tPA antibody during in vitro activation by the calcium ionophore inhibited the activation-induced zona hardening and also preserved the ability of the oocyte to be penetrated by sperm subsequent to activation. Rat oocytes undergo zona hardening during in vitro maturation in the absence of serum, presumably as a result of spontaneous cortical granule release, based on findings in mice and hamsters. In the present study, the anti-tPA antibody prevented the zona hardening and enhanced partition by spermatozoa of rat oocytes that were matured in vitro without serum. Collectively, the observations reported have suggest a possible role of tPA released during the cortical granule reaction in the zona reaction, which contributes to the block to polyspermy.     

Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes.

Complexes between 125I-labeled urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) bound to purified alpha 2-macroglobulin (alpha 2M) receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP). No binding was observed when using uPA. The magnitude of uPA.PAI-1 binding was comparable with that of the alpha 2MR-associated protein (alpha 2MRAP). Binding of uPA.PAI-1 was blocked by natural and recombinant alpha 2MRAP, and about 80% inhibited by complexes between tissue-type plasminogen activator (tPA) and PAI-1, and by a monoclonal anti-PAI-1 antibody. In human monocytes, uPA.PAI-1, like uPA and its amino-terminal fragment, bound to the urokinase receptor (uPAR). Degradation of uPAR-bound 125I-uPA.PAI-1 was 3-4-fold enhanced as compared with uncomplexed uPAR-bound uPA. The inhibitor-enhanced uPA degradation was blocked by r alpha 2MRAP and inhibited by polyclonal anti-alpha 2MR/LRP antibodies. This is taken as evidence for mediation of internalization and degradation of uPAR-bound uPA.PAI-1 by alpha 2MR/LRP.     

A monoclonal antibody directed against the high-affinity lysine-binding site (LBS) of human plasminogen. Role of LBS in the regulation of fibrinolysis

One of thirty murine monoclonal antibodies, raised by immunization with human plasmin-alpha 2-antiplasmin complex, was found to be directed against the high-affinity lysine-binding site in plasminogen. Indeed, this antibody (MA-HAL) reacted with plasminogen and with a fragment of plasminogen composed of the first three triple-loop structures (LBS I) and was displaced by 6-aminohexanoic acid (50% displacement at 25 microM). In competitive radioimmunoassays the binding of radiolabeled plasminogen to MA-HAL was reduced to 50% with 2.3 microM alpha 2-antiplasmin or 1.3 microM histidine-rich glycoprotein, which corresponds to the known dissociation constants between these ligands and the high-affinity lysine-binding site of plasminogen. MA-HAL did not influence the activation of plasminogen by tissue-type plasminogen activator in the absence of CNBr-digested fibrinogen, but abolished the effect of CNBr-digested fibrinogen on the Michaelis constant of the reaction. MA-HAL reduced the reaction rate between plasmin and alpha 2-antiplasmin by a factor 20 and abolished the binding of plasminogen to fibrin. These results indicate that MA-HAL specifically binds to and masks the high-affinity lysine-binding site of plasminogen. It therefore is a useful tool for the investigation of the role of this structure in the regulation of fibrinolysis, both at the level of fibrin-stimulated activation of plasminogen and of the inhibition of generated plasmin.     

Mechanism of inhibitory effect of angiostatin on plasminogen activation by its physiologic activators

The influence of angiostatin K1-4.5--a fragment of the heavy chain of plasmin and a powerful inhibitor of angiogenesis--on kinetic parameters (k(Pg) and K(Pg)) of human Glu-plasminogen activation under the action of urokinase (uPA) not having affinity for fibrin and fibrin-specific tissue plasminogen activator (tPA) was investigated. Angiostatin does not affect the k(Pg) value, but increases the value K(Pg) urokinase plasminogen activation. A decrease in the k(Pg) value and an increase in the K(Pg) value were found for fibrin-stimulated plasminogen activation by tPA with increasing concentrations of angiostatin. The obtained results show that angiostatin is competitive inhibitor of the uPA activator activity, while it inhibits the activator activity of tPA by mixed type. Such an influence ofangiostatin on the kinetic constants ofthe urokinase plasminogen activation suggests that angiostatin dose dependent manner replaces plasminogen in the binary enzyme-substrate complex uPA-Pg. In case of fibrin-stimulated plasminogen activation by tPA, both zymogen and tPA are bound to fibrin with formation of the effective triple tPA-Pg-fibrin complex. Angiostatin replaces plasminogen both from the fibrin surface and from the enzyme-substrate tPA-Pg complex that leads to a decrease in k(Pg) and an increase in K(Pg) of plasminogen activation. Inhibition constants by angioststin (Ki) of plasminogen-activator activities of uPA and tPA determined by Dixon method were found to be 0.59 +/- 0.04 and 0.12 +/- 0.05 microM, respectively.     

Characterization of 125I-tissue plasminogen activator binding to cerebellar granule neurons

Mouse cerebellar cells in culture secrete tissue plasminogen activator (tPA) into the culture medium. Fibrin overlays have shown tPA to be associated with granule neurons in these cultures. This cell associated tPA can be displaced by extensive washing of the cells or by a brief lowering of the pH (less than 4), which leads to a loss of fibrinolytic activity by the cells. Incubation of these fibrinolytically inactive cells with exogenously added murine tPA leads to the restoration of the fibrinolytic activity, indicating the presence of tPA binding sites on these granule neurons. Using 125I-tPA, the binding to cerebellar granule neurons is rapid, saturable, specific, high affinity (Kd = 50 pM) and reversible. Both murine and human tPA compete with 125I-tPA for binding, with both murine and human urokinase (uPA) as well as human thrombin and plasminogen fail to compete. Neither the catalytic site nor the carbohydrate moiety of tPA appear to be involved in the binding, since both diisopropyl-fluorophosphate-treated tPA and endoglycosidase-H-treated tPA compete with 12I-tPA for binding. Furthermore, epidermal growth factor does not compete well with tPA for binding even at a 10:1 molar excess, suggesting that the epidermal growth factor-like (EGF) domain of tPA may not be involved in the binding mechanism. Autoradiography and antibody immunofluorescence show the specific tPA binding is to granule neurons in these cultures. Thus, granule neurons possess tPA receptors on their surface, where this protease binds retaining is functional activity and may play a role in cell and axon migration.