doi:10.1046/j.1432-1033.2003.03549.x
Eur. J. Biochem. 270, 1850–1854 (2003) (cid:3) FEBS 2003
Tetranectin binds hepatocyte growth factor and tissue-type plasminogen activator
Uffe B. Westergaard, Mikkel H. Andersen, Christian W. Heegaard, Sergey N. Fedosov and Torben E. Petersen
Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Denmark
analysis of the tPA-catalysed plasminogen activation was performed. The kinetic parameters of the tetranectin- stimulated enhancement of tPA were comparable to fibri- nogen fragments, which are so far the best inducer of tPA-catalysed plasminogen activation. The enhanced activation was suggested to be caused by tetranectin’s ability to bind and accumulate tPA in an active conformation.
Keywords: tetranectin; plasminogen; hepatocyte growth factor; tissue-type plasminogen activator.
In the search for new ligands for the plasminogen kringle 4 binding-protein tetranectin, it has been found by ligand blot analysis and ELISA that tetranectin specifically bound to the plasminogen-like hepatocyte growth factor and tissue-type plasminogen activator. The dissociation constants of these complexes were found to be within the same order of mag- nitude as the one for the plasminogen-tetranectin complex. The study also revealed that tetranectin did not interact with the kindred proteins: macrophage-stimulating protein, urokinase-type plasminogen activator and prothrombin. In order to examine the function of tetranectin, a kinetic
Tetranectin (TN) is a homotrimeric C-type lectin [1]. It was originally purified due to its specific affinity towards the kringle-4 domain of plasminogen (Plg) [2]. Each of the three 20 kDa-monomers consists of an N-terminal heparin-bind- ing domain, an a-helical domain involved in the trimeriza- tion through a triple coiled coil structure, and a C-terminal carbohydrate recognition domain responsible for the bind- ing to Plg [3,4]. Interaction between the carbohydrate recognition domain and Plg is both lysine and calcium sensitive, each of which practically abolished the binding between TN and Plg [3].
Although the biological function of TN is still uncertain, there has been some speculation. One suggestion is that TN forms a link between the extracellular matrix and Plg by linking Plg to sulphated polysaccharides, thus enabling a local tissue remodelling. Therefore, TN may be involved in events leading to the proteolysis of matrix proteins, as activated Plg is believed to play a key role in the degradation of the extracellular matrix. Another possibility is that TN stimulates mineralization during osteogenesis [11] and may participate in myogenesis during embryonic development as well as muscle regeneration [12]. Thus, the TN-employing mechanisms are likely to be generally applicable to tissue remodelling and not just a characteristic feature of tumour invasion.
At the present stage TN has been reported to bind apolipoprotein(a) [13], fibrin [14], Plg, and some sulphated polysaccharides [15]. Here evidence is presented of two novel TN-binding proteins: hepatocyte growth factor (HGF) and tissue-type plasminogen activator (tPA), whereas three related proteins: macrophage-stimulating protein (MSP), urokinase-type plasminogen activator (uPA), and pro- thrombin, appeared to be incapable of TN-binding. TN was originally isolated from plasma [2] but it shows a wide tissue distribution. Predominantly, TN was found in the secretory cells of endocrine tissue like pituitary, thyroid, parathyroid glands, and the liver, pancreas, and adrenal medulla [5]. A distinct accumulation of TN was observed in the surrounding extracellular matrix of various carcinomas [6–8] where it colocalized with Plg [9]. Whether the TN originated from plasma or it was produced by the surround- ing tissue is still unknown. However, it has been established that the TN concentration in plasma decreased parallel with the growth of the tumour and this characteristic is considered to be an indication of poor survival prognosis [10].
Experimental procedures
Correspondence to T. E. Petersen, Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Gustav Wieds Vej10 C, DK-8000 Aarhus C, Denmark. Fax: + 45 86 13 65 97, Tel.: + 45 89 42 50 94, E-mail: tep@mbio.aau.dk Abbreviations: TN, tetranectin; Plg, plasminogen; HGF, hepatocyte growth factor; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator; MSP, macrophage- stimulating protein. (Received 26 July 2002, revised 4 February 2003, accepted 28 February 2003)
Expression and purification of recombinant tetranectin
Using the previously cloned TN cDNA [16] from a murine lung cDNA library as template in a polymerase chain reaction the coding sequence of murine TN was amplified and inserted into pPICZa-vector (Invitrogen, Netherlands) containing a signal peptide, a myc-epitope for tracing, and six histidine residues for purification at the N-terminal end of the expressed protein. Additionally, the sequence of a coagulation factor Xa cleaving site (Ile-Glu-Gly-ArgflGly) was inserted at the 5¢-end of the TN-sequence to facilitate
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cleavage of the N-terminal service peptides (myc, His6) after protein purification. The construct was transfected into the protease-deficient SMD1168 strain of Pichia pastoris as described by the manufacturer.
1 mM EDTA buffer between every incubation. There was an additional step in the case of HGF when the antihuman HGF-containing wells were incubated with 100 lL of 1 lgÆmL)1 human HGF for 2 h at 37 (cid:4)C. All incubations were performed in blocking buffers. The compounds immobilized in the wells were then exposed to TN, which concentration varied from 0 to 50 lgÆmL)1 in 100 lL. The incubation continued for 2 h at 37 (cid:4)C. After washing, the wells were treated with 100 lL custom-made (DAKO, Roskilde, DK), specificity-checked rabbit anti-murine TN serum (1 : 1000) for 1 h at 37 (cid:4)C. Then 100 lL of peroxidase-conjugated porcine anti-rabbit IgG (1 : 2000) was added and incubated for one hour at 37 (cid:4)C. Finally, the TN-positive samples were visualized by a coloured reaction with o-phenylenediamine. The reaction was stopped with 100 lL 2 M H2SO4, and the absorbance at 490 nm gave a relative content of bound TN.
Plasminogen activation assay
Positive clones were initially grown in shake flasks on glycerol to gain a high cell density. Afterwards, the carbon source was changed to 0.5% methanol, which induced expression of TN. The fermentation was carried out as outlined by the manufacturer. The cell free culture medium was saturated with ammonium sulphate to 95% and centrifuged. The precipitate was dissolved in 50 mM Na2HPO4 pH 7.4, 0.14 mM NaCl and dialyzed overnight at 4 (cid:4)C to decrease the salt concentration. From preliminary experiments, the sample was expected to contain a mixture of two TN-forms: myc-His6-TN and TN without the service peptides (the latter one to be prevailing). Therefore, the myc- His6-TN form was separated from the other by adsorption on a Ni-matrix, and TN was purified as described below. The dialyzed sample was mixed with Ni-NTA-Sepharose, loaded into a column, and washed with 50 mM Na2HPO4 pH 7.4, 0.5 M NaCl. The run through and the wash fractions were collected and dialyzed overnight at 4 (cid:4)C to remove salt. The obtained preparations were adsorbed on an S-Sepharose column and washed until a stable baseline was reached. TN was eluted with 50 mM Na2HPO4 pH 7.4, 0.5 M NaCl and concentrated by ultrafiltration.
sequencing showed the
The identity of the purified protein was confirmed by N-terminal sequence and GESPTPKAKK…, which compared to the native sequence (ESPTPKAKK…) only including an additional N-terminal glycine. The concentration of TN stocks was determined by amino acid analysis.
The ability of TN to enhance the plasminogen activation potential of tPA was investigated in a coupled reaction assay by measuring hydrolysis of the plasmin substrate S- 2251 (H-D-Val-Leu-Lys-p-nitroaniline). The proteolytic cleavage of the substrate resulting in release of the yellow p-nitroaniline was followed spectroscopically at 405 nm. The plasminogen activation assay was carried out in 100 lL 0.1 M Tris pH 7.4, 0.02% Tween 80, 5 lM TN, and 0.5 mM S-2251 at varying concentrations of bovine plasminogen (0.1–2.0 lM). The reaction was initiated by addition of 10 nM human tPA, and the appearing plasmin activity was followed for 3 min at 37 (cid:4)C. Two independent experiments were made for each Plg con- centration. Ligand blot analysis using 125I-labelled tetranectin
K1
The activation of Plg and the subsequent hydrolysis of the plasmin substrate can be described in the simplest case by the following scheme:
k1 tPA þ Pln
tPA þ Plg ! tPA-Plg (cid:4)! ð1Þ
K2 Pln-S (cid:4)!
k2 Pln þ P
Pln þ S ! ð2Þ
(kcat). The process was
reaction. The amidolytic activity of the
where Pln represents plasmin, S and P represent the plasmin substrate S-2251 and the yellowish product P, respectively, K1 and K2 are the Michaelis constants (Km) of the corresponding enzymes, k1 and k2 are their catalytic constants carried out at [tPA] << [Plg] and [Plg] (cid:7) constant during the time of tPA (tPA + S fi tPA + P) can be ignored because of its low efficiency when compared with Pln. Three micrograms of bovine Plg, 4 lg human tPA, 2 lg human uPA, 2 lg human HGF (294-HGN, R&D Systems Europe, UK), 3 lg bovine prothrombin, and 2 lg human MSP (352-MS, R&D Systems) were dissolved in the Laemmli-buffer containing 2% SDS and subjected to SDS/PAGE. Subsequent electroblotting was carried out on Immobilon-P transfer membranes (Millipore, Bedford, MA, USA). The membrane was blocked with 5% BSA, 0.05% Tween 20 and 1 mM EDTA in 50 mM Na2HPO4 pH 7.4 for 2 h. The blot was then incubated overnight 4 (cid:4)C with 125I-labelled TN corresponding at to 100 000 cpmÆmL)1. TN was labelled with 125I according to the chloramine T method. After washing 3 · 15 min with 0.05% Tween 20 and 1 mM EDTA in 50 mM Na2HPO4 pH 7.4 the blot was dried and visualized by autoradiography.
The collected data (t,p) was transformed and analyzed according to a previously published model [17] that gives a linear dependence of y on t2
y ¼ y0 þ vat2
(cid:1) ln S0ep=K2 =ðS0 (cid:4) pÞ
(cid:2) and y0 is the error in where y ¼ 2 K2 k2 determination of the zero point (y0 (cid:7) 0). The transforma- tion of P to y was carried out with the known values of K2 ¼ 250 lM and k2 ¼ 1000 min)1 [18] and the substrate concentration from this experiment s0 ¼ 500 lM. The slope Concentration-dependent binding assays ELISA-trays (96-well) were coated with 100 lL 2 lgÆmL)1 bovine Plg, human tPA, human uPA, bovine prothrombin, or monoclonal anti-human HGF (MAB694, R&D Sys- tems) at 4 (cid:4)C overnight. The wells were then blocked for 1 h at 37 (cid:4)C with 200 lL 0.5% gelatine and 1 mM EDTA dissolved in 50 mM Na2HPO4 pH 7.4, 0.14 mM NaCl. The wells were washed briefly three times with 200 lL of 50 mM Na2HPO4 pH 7.4, 0.14 mM NaCl, 0.05% Tween 20,
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va is equal to the velocity at a given [Plg]. A number of ([Plg],va) pairs were obtained and fitted to the equation va ¼ ðk1½tPA(cid:10)0½Plg(cid:10)Þ=ðK1 þ ½Plg(cid:10)Þ which enabled calcula- tion of the kinetic parameters Km and kcat of tPA towards Plg, see [17] for details. The ligand blot analysis (Fig. 1) revealed binding of TN to Plg, tPA, and HGF, whereas uPA, prothrombin, and MSP showed no binding properties towards TN. Distinct bands of Plg and HGF were visible after a short time of exposure. The tPA band was somewhat weaker, but it became clearer after longer exposure.
Results
Concentration-dependent binding assays Purification of the recombinant tetranectin
To validate the results from the ligand blot analysis the interactions were tested by ELISA. The solid-phase binding assays revealed a concentration-dependent binding of TN to Plg, HGF, and tPA. As expected, no specific binding was observed for uPA and prothrombin (Fig. 2). MSP was not included in this assay. Table 1 presents the summarized data from the ELISA, where the specific affinities are compared to unspecific binding, and the measured dissociation constants Kd are listed for Plg, HGF, and tPA.
The recombinant TN was expressed as a mixture of two forms (myc-His6-TN and TN) due to occasional N-terminal cleavage of the protein at the Xa-site in the yeast. The form without service peptides (TN) prevailed and it was therefore separated from myc-His6-TN by absorption of the latter on Ni-NTA-sepharose. The run through fraction was subjected to S-Sepharose, which enabled TN purification. Two intense bands each with a relative molecular mass of approximately 20 kDa were visualized on SDS/PAGE (Fig. 1). These bands corresponded to TN on Western blot (data not shown). Amino acid sequencing of the N-terminus revealed that the myc-epitope and the histidine-tag were cleaved off at the coagulation factor Xa-site upon secretion leaving the recombinant TN of the desired length. An additional cleavage occurred after Lys10 as well. From N-terminal sequence analysis it was estimated that the purified protein contained a 1 : 4 mixture of full-length and N-terminally cleaved TN.
Fig. 2. Concentration-dependent binding of ligands to TN. The curves correspond to plasminogen (s), tPA (n), HGF (e), uPA (h), and prothrombin (,). Each point is the mean of quadruplicate determi- nations. In the case of uPA and prothrombin, no Kd could be deter- mined.
Ligand blot analysis
Table 1. Summary of TN affinity assays. The column (cid:2)Bound TN(cid:3) corresponds to the amount of TN detected in the wells coated with a potential ligand; Unspecific binding corresponds to the amount of TN detected in the uncoated wells. The percentages are standardized according to plasminogen (100%). Calculation of the Kd is based on Michaelis–Menten kinetics. Dissociation constants for the binding of TN to plasminogen and tPA are based on three independent experi- ments of quadruplicate measurements. For the TN-HGF binding, the Kd is based on one experiment of quadruplicate measurements. In the case of uPA and prothrombin, one and two independent experiments of quadruplicate measurements were performed, respectively.
Bound TN (%)
Unspecific binding (%)
Kd (lM)
100.0 ± 9.5 84.3 ± 8.7 97.2 ± 14.5 14.2 ± 0.4 17.4 ± 5.8
6.2 ± 3.2 11.8 ± 0.5 8.7 ± 4.7 9.0 ± 1.0 8.1 ± 1.1
0.33 ± 0.05 0.49 0.28 ± 0.09 – –
Plg HGF tPA uPA Prothrombin
Fig. 1. SDS/PAGE/Ligand blot analysis. The samples were reduced prior to electrophoresis. Left: SDS/PAGE indicates two bands of TN as a result of N-terminal cleavage. Right: Lanes 1–6 were loaded with plasminogen, tPA, uPA, HGF, prothrombin, and MSP, respectively. The blot shows TN-binding to plasminogen and HGF. However, longer exposure revealed binding to tPA as well.
In an attempt to identify new compounds capable of TN- binding, several candidates were subjected to ligand blot analysis. The proteins were chosen in the light of their contents of kringle domains. Plg with five kringle domains served as a positive control for the binding of TN. The plasminogen-like growth factors HGF and MSP are very similar to Plg in their domain structure and each of them contains four kringle domains. Prothrombin has two kringle domains and the structures of plasminogen activators tPA and uPA include two and one kringles, respectively.
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Two different experiments complemented and supported each other, thus confirming the affinities of the novel TN-binding proteins.
Plasminogen activation assay
(0.28 ± 0.09 lM) are within the same order of magnitude pointing to the same type of binding for all three ligands. In other words, the carbohydrate recognition domain is very likely to be responsible for the binding of HGF and tPA, although this needs further clarification. Differences in Kd are insignificant and may be caused by the nature of the primary and secondary antibodies used for detection.
It was found in the preliminary experiments that the amidolytic activities of TN-Pln and TN-tPA complexes towards the substrate S-2251 were equal to those of Pln and tPA and that no difference was found in the uPA-mediated activation of Plg with or without the inclusion of TN. This validates the direct application of Eqn (1) for calculation of va at different Plg-concentrations. The final data are presented in Fig. 3 as a plot of the reaction velocity va vs. the concentration of Plg. The analysis reveals the kinetic parameters of the activation of Plg by tPA in the presence of TN: Km ¼ 0.28 lM and kcat ¼ 10 min)1. In the control experiment the kcat could not be determined. However, when approximating kcat to 10 min)1, Km can be calculated to 2.5 lM.
Discussion
The results presented here show evidence for TN-binding to HGF and tPA by two independent methods, thereby adding two kringle-containing proteins to the list of TN-ligands. The affinity of the recombinant TN to Plg (comparable to the one of the natural TN) demonstrates that the recombinant protein has been correctly folded in P. pastoris and hence is relevant for the affinity assays. Lack of interaction between TN and uPA indicates that TN does not have a general affinity towards kringle domains. Moreover, TN does not bind MSP, a growth factor structurally similar to HGF. The for Plg’s binding calculated Kd
It has long been known that TN stimulates the activating cleavage of Plg by tPA, though, no details about the process have been known [2]. The present study verifies the activating ability of TN and shows that the presence of TN increases the association between tPA and Plg by 10-fold. The values of Km and kcat calculated for the TN–tPA complex were similar to the parameters for the tPA activation of Plg in the presence of fibrinogen fragments (Km ¼ 0.1 lM and kcat ¼ 25 min)1) [17]. This implies that TN may act in the same way as fibrinogen fragments. The experimental data were, however, not conclusive concerning the exact mechanism behind the enhancement. Recently, we have reported that bovine tPA exists in equilibrium between two different conformations, where only a minor part of tPA-molecules can bind and cleave Plg. Fibrinogen frag- ments bind only the active form of tPA and thereby poises the equilibrium towards accumulation of the active confor- mation. The net result is an increased concentration of active tPA capable of cleaving Plg [17]. An analogous mechanism was suggested for human tPA, though, conver- sion between inactive and active tPA occurred rapidly and was difficult to detect by conventional measurements. It seems possible that the TN-induced activation of tPA is similar to the one of fibrinogen fragments. Another explanation suggests that TN is able to bind the active form of tPA and Plg simultaneously and by bringing the it acts as a cofactor in the two components together, activation of Plg to plasmin. The ability of TN to accumulate by one or another mechanism the active tPA in the extracellular matrix enables a higher control of the local plasmin activity. to TN (0.33 ± 0.05 lM) is consistent with the previously pub- lished dissociation constants of 0.5 lM and 0.2 lM [3,13]. The values of Kd for HGF (0.49 lM) and tPA
Fig. 3. tPA-catalysed plasminogen activation in the presence of TN. The curve represents a plot of the reaction velocity va vs. the concentration of plasminogen fitted to a Michaelis equation. Data obtained in the presence of TN (s) and control data without TN (d) are based on two or more measurements. The kinetic analysis revealed that TN has a resemblance to fibrinogen fragments in respect to the enhancement of tPA-catalysed plasminogen activation.
HGF is a mitogen for a variety of cells including epithelial and endothelial cells, melanocytes, keratinocytes, and hepatocytes. Additionally, the cytokine stimulates cell motility and morphogenesis. HGF has also been shown to take part in angiogenesis. HGF is a 90-kDa glycoprotein composed of a 60-kDa a chain and a 30-kDa b chain covalently linked by a disulphide bond. The a chain contains an N-terminal heparin-binding hairpin-loop and four kringle domains. The b chain is homologous to serine proteases, but lacks proteolytic activity due to mutations in the catalytic triad. HGF is secreted as a biologically inactive single-chain form and extracellular processing is required to obtain the active two-chain HGF. This is accomplished by a specific cleavage at Arg494-Val495, similar to the Arg-Val cleavage required for activation of Plg to plasmin [19].
There is evidence that single-chain HGF can be activated along two or more pathways. In response to a tissue injury, epithelial cells produce HGF activator, a 34-kDa serine protease, that once it has been activated by thrombin, can process single-chain HGF [20]. Another pathway involves the plasminogen activators tPA and uPA. It has been shown in in vitro bioassays that tPA and uPA can convert single- chain HGF into biologically active HGF [21]. Taking the activation of HGF by tPA together with the new findings
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(1996) Tetranectin and plasmin/plasminogen are similarly distri- buted at the invasive front of cutaneous melanoma lesions. J. Pathol. 179, 260–265.
10. Høgdall, C.K., So¨ le´ tormos, G., Nielsen, D., Nørgaard-Pedersen, B., Dombernowsky, P. & Clemmensen, I. (1993) Prognostic value of serum tetranectin in patients with metastatic breast cancer. Acta Oncol. 32, 631–636.
11. Wewer, U.M., Ibaraki, K., Schjørring, P., Durkin, M.E., Young, M.F. & Albrechtsen, R. (1994) A potential role for tetranectin in mineralization during osteogenesis. J. Cell Biol. 127, 1767–1775.
that TN can both bind and enhance tPA activity, it is tempting to hypothesize whether TN is involved in the regulation of HGF as well. Testing this hypothesis would seem evident. However, several unsuccessful attempts were made to express recombinant single-chain HGF as the commercially available HGF is in an already activated form. The colocalized expression of HGF and tPA in the murine olfactory system tells in favour of this suggestion [22].
Acknowledgements
This work was supported by a grant from Novo Nordisk A/S with a scholarship to UBW.
12. Wewer, U.M., Iba, K., Durkin, M.E., Nielsen, F.C., Loechel, F., Gilpin, B.J., Kuang, W., Engvall, E. & Albrechtsen, R. (1998) Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro. Dev. Biol. 200, 247–259.
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