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THE BINDING OF PLASMINOGEN FRAGMENTS TO CULTURED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS Hua-Lin Wu, Ing-Shiang Wu, Ro-Yun Fang, Jen-Shau Hau, Dung-Ho Bi-lng Chang#, Tsun-Mei Lit-@, and Guey-Yueh Shi*
Wu,
Departments of Biochemistry and #Medical Technology, Medical College, National Cheng Kung University, Tainan, Taiwan 700, Republic of China Received
August
25,
1992
Glu-plasminogen, kringle l-5, kringle l-3; and miniplasminogen exhibited strong binding to human umbilical vein endothelial cells (HUVEC). On the other hand, no significant binding was obtained with microplasminogen and kringle 4. Kringle l-5 and miniplasminogen, which both contained kringle 5, specifically inhibited the binding of plasminogen to HUVEC while kringle l-3 did not. The results implied plasminogen molecule contained at least two binding sites, with which it interacted HUVEC. The stronger binding site was located in kringle 5 and the weaker one was in kringle 1-3. Kringle 4 and the active site domain exhibited no significant binding to HUVEC. The interaction of plasminogen with HUVEC 0 1992Academic Press,Inc. is mainly through binding site on kringle 5.
Vascular endothelial cells play an important role in the regulation of the human fibrinolytic system (1). Plasminogen activators and plasminogen activator-inhibitor are synthesized and secreted by endothelial cells (2-6). Furthermore, endothelial cells provide binding sites for plasminogen and tissue-type plasminogen activator (7-10). The binding of plasminogen to endothelial cell surface enhances its activation by tissue-type plasminogen activator (11). The interaction of plasmin with endothelial cells might also be important in the feed back regulation *To whom correspondence should be addressed. The abbreviations used are: Glu-Pig, native plasminogen consisting of Glul-Asn7so; Mini-Plg, Micro-Plg, kringle 1-5, kringle l-3, and kringle 4 corresponding to plasminogen fragments consisting of Va1442-Asn7sop Lysssc-Asn790, Lys77-Arg529, Lys77-Valss7, and Vals54-Ala439, respectively; SDS, sodium dodecyl sulfate; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline; BSA, bovine serum albumin. 0006-291X/92
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of fibrinolytic activity of the cells (12). It has been demonstrated that lipoprotein(a) binds to the plasminogen binding site on endothelial cells (13, 14) and may thereby inhibit plasminogen activation by competing for the same binding site. Clinical studies also demonstrate that patients with elevated lipoprotein(a) levels have less fibrinolytic activity (15). Lipoprotein(a) contains a lipid core, an apoprotein B and an apoprotein(a) subunits (16). Protein and cDNA sequence analysis indicate a high degree of homology between apoprotein(a) and plasminogen (17). The apoprotein(a) subunit contains up to 37 copies of kringle 4 and a single copy of kringle 5, with 75-85% and 95% homology, respectively, to their plasminogen counterparts (17). Lipoprotein(a) inhibited the binding of plasminogen form 2 to monocytoid U937 cells as well as to HUVEC (14). In addition, inhibition of [1251]-plasminogen form 2 to U937 cells was blocked by monoclonal antibodies against kringle 4 (18). Thus, kringle 4 was suggested to be responsible for the binding of lipoprotein(a) to U937 Miles et al reported that lipoprotein(a) inhibited the binding of cells. [12sl]-labeled plasminogen at the same extent as that of unlabeled plasminogen (14). However, isolated kringle 4 was 100 to 300 times less potent an inhibitor of plasminogen binding to U937 and to HUVEC than lipoprotein(a) although it has similar conformation, immunochemical and functional properties as integrated kringle 4 (14). Therefore, it is suggested that the capacity of lipoprotein(a) to compete with plasminogen for cellular binding must reside in concatenation of kringle 4-like structures with lipoprotein(a). The binding can also be attributable to other homologies between the molecules such as the kringle 5 and the light-chain domain of lipoprotein(a) which is 94% homologous to the plasminogen proteinase domain (14, 19). The result obtained using platelets as models demonstrates that kringle l-3 is a primary recognition site for plasminogen binding to both thrombin-stimulated and non-stimulated platelets (20). In addition, kringle 4 and Mini-Plg, which contains kringle 5 and proteinase domains, can also participate in the interaction (20). Therefore, it is still uncertain which domain of either lipoprotein(a) or plasminogen is responsible for their binding to HUVEC. Plasminogen fragments of different sizes have been isolated by partial digestion of plasminogen with elastase and plasmin to form Mini-Plg, kringle l-3, kringle 4, Micro-Plg, and kringle 1-5 (21-25). Human plasminogen and its fragments were applied to study the interaction of plasminogen with fibrin (25). The results indicated that there are two fibrin-binding domains in plasminogen; the one in kringle 5 is of higher affinity and the other in kringle 1-3 is of lower affinity (25). The present study was designed to investigate which domain of plasminogen was 704
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Methods
Preoaration of olasminocen and olasmin: Human plasminogen was prepared from pooled human plasma by a modification of the Deutsch and Mertz method (26). Forms 1 and 2 of native plasminogen were separated by chromatography on Lys-Sepharose (27). For this study, form 2 of human plasminogen was used exclusively. Urokinase-free human plasmin was prepared by activation of human plasminogen with urokinase-Sepharose as described in a previous report (22). Preoaration of Mini-Plo. Micro-Plc. krinole 1-5. krinale l-3. a d mole 4: Mini-Plg, kringle 1-3, and kringle 4 were obtained by dige?sting Glu-Plg with a catalytic amount of porcine pancreatic elastase and purified by Sephadex G-75 (2.5 x 70 cm) and Lys-Sepharose (2 x 15 cm) columns (20, 24, 27). Micro-Plg and kringle l-5 were made by incubating Glu-Pig with plasmin in an alkaline solution and purified by Lys- and soybean-trypsin-inhibitor-Sepharoses as in the previous reports (24, 25, 28). Protein concentration: The protein spectrophotometrically using the following molecular weights, respectively: Glu-Pig, Plg, 16.0 and 38,000 (21); Micro-Plg, 16.0 17.0 and 55,000 (25). The E 28onm values 4 were 8.08 x 104 M-‘cm-1 and 3.1 x 104
concentrations were determined &I%, 280 nm values and 17.0 and 92,000 (29, 30); Miniand 28, 617 (24); kringle l-5, used for kringle l-3 and kringle M-‘cm-1, respectively (31).
[ZI$ labeling of protein: 100 ul of protein sample at a concentration of 1 .O mg/ml was incubated with Nat251 (100 uCi) and lightly agitated at room temperature in an Eppendorf tube previously coated with 100 1.11of lodo-gen (0.08 mg/ml) for 8 min. After incubation, the reaction mixture was applied to a PD-10 column and eluted with 0.01 M phosphate buffer, pH 7.2. The labeled protein was collected after The specific radioactivities varied from 2 x discarding the void volume. 10s cpm to 5 x 10s cpm/mg of protein. Cell culture: Endothelial cells were isolated from human umbilical cord veins by reaction with collagenase following the previously described method (32). Cells were grown to confluence in medium M-199 containing 10% fetal calf serum, 25 ug/ml endothelial cell growth factor, 10 units/ml heparin, 100 units/ml penicillin, and 100 units/ml Passaged cells were subcultured in 96-well dishes and streptomycin. allowed to grow for 3 days to confluence under the same condition as Third passaged cultures were employed in all primary cultures. experiments. The cell count at confluency averaged 3 x 104 per well. Prior to using live cells in binding studies, the confluent cell culture was washed 5 times with 0.1 M PBS, pH 7.4 containing 0.25% BSA. 705
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hvde-fixed c&: The HUVEC monolayer was washed 5 times with M-199, once in 0.1 M PBS, pH 7.4 at 37OC and then fixed in 0.02% (v/v) glutaraldehyde in PBS at 37X for 20 min as described (11). Fixed cells were washed 3 times with PBS-Tween 20-BSA (0.1 M PBS, pH 7.4 containing 0.5% Tween 20 and 0.1% BSA) and passivated 60 min in the same buffer. Binding studies were performed after wash fixed cell monolayers 3 times with 0.1 M PBS, pH 7.4 containing 0.25% BSA. Bindina of olasminoaen fraaments to HUVEC: Measurement of binding of [1251]-Giu-Pig to HUVEC was carried out using live and fixed endothelial cell monolayers. The binding of plasminogen fragments such as Mini-Plg, Micro-Plg, kringle l-5, kringle 1-3, or kringle 4 to HUVEC was done using fixed cells. [125l]-labeled protein at various concentrations in 100 ~1 PBS containing 0.25% BSA and 0.1 p.M aprotinin, with or without 10 mM tranexamic acid were added to triplicate tissue culture wells containing live or fixed cell monolayers. The tissue culture plates were incubated at 4OC for 30 min and 50-pl aliquots of the culture supernatant were removed from the wells and counted in an LKB y-counter (value A). The wells were then washed 5 times with PBS (total wash time 45 s) and the cells in each well were solubilized in 150 p.1 of 1% SDS, 0.5 M NaOH and 0.01 M EDTA (11). The radioactivities of total cell lysates were measured with a y-counter (value B). The amount of protein bound to HUVEC per well = the amount of protein added x B /(2A + B). Specific binding was calculated as binding in the absence of tranexamic acid (total binding) minus binding in the presence of tranexamic acid (nonspecific binding). The molecules of ligand bound per cell were calculated from the specific binding of each protein. Cell number was determined by trypsinization of the cells from wells on each 96-well tissue culture plates and by counting the cells under a microscope. SDS oolvacrvlamide ael electroohoresis: The basic techniques electrophoresis was done as described by Weber and Osborn (33).
of gel
Reagents: lodo-gen, BSA. tranexamic acid and elastase were purchased from Sigma. Sepharose 48 and PD-10 were from Pharmacia LKB Biotechnology. Endothelial cell growth factor was from Collaborative Incorporated. Medium M-199, fetal calf serum, penicillin and streptomycin were from Gibco. All other reagents used were analytical grade. Results The binding of human Glu-Pig to HUVEC was a concentration-dependent reaction. Pretreatment of HUVEC with glutaraldehyde did not significantly alter the binding of Glu-Pig to the cells (Fig. 1). The endothelial cells pretreated with glutaraldehyde were used for binding assays, since less cell detachment was observed and more consistent 706
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Added
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125 I-Glu-PlglTotal
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Fig 1. Binding of [tW]-Glu-Plg to HUVEC. Fixed and living HUVEC monolayers were incubated with [ 12sl]-Glu-Pig for 30 min at 4% in the presence or absence of 10 mM tranexamic acid. The total binding ( o), nonspecific binding ( l ), specific binding (m) of fixed cells, and the specific binding (A) of living cells were determined as described under “Materials and Methods”. Each data point represents the mean of triplicate well experiments. Fig. 2. Effect of unlabeled Glu-Pig on the binding of [12sl]-Glu-Pig to HUVEC. Fixed HUVEC were incubated with 0.1 to 1 uM of labeled Glu-Pig and 0.9 to 0 PM unlabeled Glu-Pig at a final total concentration of I PM for 30 min at 4% in the presence or absence of 10 mM tranexamic acid. Specific binding was determined by subtracting “nonspecific” (nontranexamic acid inhibitable) binding from total binding at each incubation. Each data point represents the mean f S.D. of triplicate samples.
results could be obtained. The binding of Glu-Plg to HUVEC was specifically inhibited by 10 mM tranexamic acid (Fig. 1). Therefore, tranexamic acid was added to the samples to obtain the values of Specific binding was calculated by subtracting nonspecific binding. “nonspecific” (non-tranexamic acid inhibitable) binding from total binding Native unlabeled Glu-Plg and [1251]-Glu-Plg at each ligand concentration. had the same efficiency in the competition for the binding to HUVEC (Fig. 2). Therefore, radioiodination of Glu-Plg did not alter the binding property. The number of molecules of ligand bound per cell at a added were approximately 2 x 107 per concentration of 1 uM [ 125l]-Glu-Pig fragments, which contained only cell (Fig. 1 and 2). Purified plasminogen one major protein band in each fraction as analyzed by SDS-gel The molecular weights of Mini-Plg, electrophoresis, were prepared. Micro-Plg, kringle 4, kringle l-3, and kringle 1-5 were 38, 28, 10, 33, and 55 KDa, respectively (Fig. 3). Mini-Plg, kringle l-5, and kringle 1-3 bound 707
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KDa -94 -07
abcdef
Fig. 3. SDS polyacrylamide gels of plasminogen fragments. Lane a:MiniPlg; b:Micro-Plg; c:Kringle 4; d:Kringle 1-3; e:Kringle 1-5; f:Glu-Plg.
to HUVEC dose-dependently (Fig. 4). However, Micro-Plg and kringle 4 did not have significant binding to HUVEC. The binding potency of plasminogen fragments to HUVEC was in the order of Glu-Pig > Mini-Plg > kringle I-5 > kringle 1-3 (Fig. 4). The ability of plasminogen fragments at various concentrations to compete with [ 1251]-Glu-Pig (1 PM) for binding to HUVEC
0.0
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Fig. 4. Binding of plasminogen fragments to HUVEC. [t2sl]-Glu-Pig ( l ), [12sl]-Mini-Plg (AL), [12sl]-kringle l-5 (o), [12sl]-kringle 1-3 ( n), [‘=I]kringle 4 ( q), or [12sl]-Micro-Plg (A) was added at increasing concentrations in the presence or absence of 10 mM tranexamic acid to determine specific binding as described in the legend to figure 2. Each data point represents the mean f S.D. of triplicate samples. Fig. 5. Inhibition of [12sl]-Glu-Plg binding to HUVEC with excess unlabeled plasminogen fragment. [lW]-Glu-Plg (1 PM) and unlabeled Glu-Plg ( l ), kringle 1-5 ( o), Mini-Plg (A), or kringle l-3 ( n) were incubated with HUVEC for 30 min at 4%. The specific binding of each incubation was determined as in the legend to figure 2. Each data point represents the mean f S. D. of triplicate samples. 708
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was studied. The binding of [ 1251]-Glu-Pig to endothelial cells was inhibited by kringle 1-5 and Mini-Plg as well as by unlabeled Glu-Pig, but not by kringle 1-3 (Fig. 5). The intact Glu-Pig was a more effective competing ligand than any of the plasminogen fragments, and the kringle 5 containing plasminogen fragments, kringle 1-5 and Mini-Plg, were also effective in inhibiting the binding of [125l]-Glu-Plg to HUVEC. Discussion Plasminogen was shown to bind to a variety of cell types including platelets, monocytes, lymphocytes, endothelial cells, smooth muscle cells, and fibroblasts (8, 20). Recent study of Miles et al indicated that a-enolase was a candidate plasminogen receptor on U937 cells (34). This study demonstrated that plasminogen specifically bound to HUVEC, and the number of Glu-Pig molecules bound per cell were 2 x 107 for HUVEC at an input Glu-Pig concentration of 1 PM. This result is comparable with those obtained in previous studies (7, 8). The results of this study indicate that the binding of plasminogen to HUVEC was mainly through kringle 5, since kringle 5containing plasminogen fragments, kringle l-5 and Mini-Plg, can effectively compete for the binding of [ f251]-Glu-Pig to HUVEC (Fig. 5). On the other hand, kringle l-3 did not inhibit the binding of [1251]-Glu-Pig to HUVEC (Fig. 5). Thus, kringle 5 should exhibit the strong binding site and kringle l-3 the weak binding site. The similar results were obtained in the studies of plasminogen binding to fibrin (25). Several studies indicated that lipoprotein(a) may modulate fibrinolysis by competing with plasminogen for binding sites on both fibrin and fibrinogen (35-37). Since binding of lipoprotein(a) to fibrin and fibrinogen increases with partial proteolysis with plasmin, lysine-binding sites are assumed to be involved in the binding of lipoprotein(a) to fibrin and fibrinogen (35). Therefore, kringle 4 and perhaps kringle 5 domains of lipoprotein(a) are suggested to be responsible for the binding of lipoprotein(a) to fibrin and fibrinogen. Results from studies using U937 monocytoid and endothelial cells demonstrate that lipoprotein(a) binds to these cells with an affinity equal to that of plasminogen (13, 18). This observation is to some extent unexpected since lipoprotein(a) has such a higher number of potential binding sites in its many repeated kringles as compared to plasminogen which contains only one copy of both kringle 4 and kringle 5. It is therefore suggested that only a few of kringle 4 present in lipoprotein(a) may be available for binding with cells, or other kringle such as kringle 5 may be responsible for the interaction. This study indicates that kringle 5 of plasminogen, not kringle 4, is responsible 709
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for the interaction of plasminogen with HUVEC. This observation is consistent with the result that krjngle 4 is less effective in inhibition of plasminogen binding to HUVEC (14). The binding of plasminogen to either fibrin or endothelial cell surface enhances its activation by plasminogen activators (11, 38-41) is also consistent with the observation that binding of plasminogen to both fibrin and endothelial cells is mainly determined by kringle 5. Whether lipoprotein(a) interacts with endothelial cells through kringle 4 or 5 is subjected to further studies. Acknowledamen&: The expert technical assistance Tsai and S. -F. Chen are gratefully acknowledged. supported by Grant NSC 80-0412-B008-80R, NSC NSC 81-0412-B006-18 from the National Science China.
of L. -C. Chang, C. -F. This work was 80-0412-B006-06, and Council, Republic of
References 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11 12.
13. 14. 15. 16.
Levin, E. G., and Loskutoff, D. J. (1982) Ann. N. Y. Acad. Sci. 401, 184194. Bachmann, F., and Kruithof, E. K. 0. (1984) Semin. Thromb. Haemost.. 10, 6-17. Van Mourik, J. A., Lawrence, D. A., and Loskutoff, D. J. (1984) J. Biol. Chem. 259, 14914-14921. Kruithof, E. K. O., Tran-Thang, C., Ransijn, A., and Bachmann, F. W. (1984) Blood 64, 907-913. Loskutoff, D. J., van Mourik, J. A., Erickson, L. A., and Lawrence, D. (1983) Proc. Natl. Sci. U.S.A. 80,6804-6808. Ramakrishnan, V., Sinicropi, D. V., Dere, R., Darbonne, W. C., Bechtol, K. B., and Baker, J. B. (1990) J. Biol. Chem. 265, 2755-2762. Miles. L. A., Levin, E. G., Plescia, J. Collen, D., and Plow, E. F. (1988) Blood 72, 628-635. Miles, L. A., and Plow, E. F. (1988) Fibrinolysis 2, 61-71. Hajjar, K., and Hamel, N. M. (1990) J. Biol. Chem. 265, 2908-2916. Russell, M. E., Quertermous, T., Declerck, P. J., Collen, D., Haber, E., and Homey, C. J. (1990) J. Biol. Chem. 265, 2569-2575. Hajjar, K. A., Harpel, E. A., Jaffe, E. A., and Nachman, R. L. (1986) J. Biol. Chem. 261, 11656-l 1662. Shi, G. -Y., Hau, J. -S., Wang, S. -J., Wu, I. -S., Chang, B. -I., Lin, M. T., Chow, Y. -H., Chang, W. -C., Wing, L. -Y. C., Jen, C. J., and Wu, H. -L. (1992) J. Biol. Chem. (in press). Hajjar, K. A., Gavish, D., Breslow, J. L., and Nachman, R. L. (1989) Nature 339, 303-305. Miles, L. A., Fless, G. M., Levin, E. G., Scanu, A. M., and Plow, E. F. (1989) Nature 339, 301-303. Hamsten, A., Walldius, G., Szamosi, A. Blomback, M., De Faire, U., Dahlen, G., Landou, C., and Wiman, B. (1987) Lancet ii, 3-9. Scanu, A. M. (1988) Arch. Pathol. Lab. Medicine 112, 1045-1047. 710
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17. McLean, J. W., Tomlinson, J. E., Kuang, W. -J., Eaton, D. L., Chen, E. Y., Fless, G. M., Scanu, A. M., and Lawn, R. M. (1987) Nature 330, 132137. 18. Gonzales-Gronow, M., Edelberg, J. M., and Pizza, S. V. (1989) Biochemistry 28, 2375-2379. 19. Edelberg, J. M., and Pizzo, S. V. (1991) Fibrinolysis 5, 135-143. 20. Miles, L. A., Dahlberg, C. M., and Plow, E. F. (1988) J. Biol. Chem. 263, 11928-l 1934. 21. Sottrup-Jensen, L., Claeys, H., Zajdel, M., Petersen, T. E., and Magnusson, S. (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191209. 22. Wu, H. -L., Shi, G. -Y., and Bender, M. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8292-8295. 23. Wu, H. -L., Shi, G. -Y., Wohl, R. C., and Bender, M. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8793-8795. 24. Shi, G. -Y., and Wu, H. -L. (1988) J. Biol. Chem. 263, 17071-17075. 25. Wu, H. -L., Chang, B. -I., Wu, D. -H., Chang, L. -C., Gong, C. -C., Lou, K. L., and Shi, G. -Y. (1990) J. Biol. Chem. 265, 19658-19664. 26. Deutsch, D. G., and Mertz, E. T. (1970) Science 170, 1095-1096. 27. Brockway, W. J., and Castellino, F. J. (1972) Arch. Biochem. Biophys. 151, 194-199. 28. Shi, G. -Y., Wu, D. -H., and Wu, H. -L. (1991) Biochem. Biophys. Res. Commun. 178, 360-368. 29. Violand, B. N., and Castellino, F. J. (1976) J. Biol. Chem. 251, 3906391 2. 30. Robbins, K. C., and Summaria, L. (1976) Methods Enzymol. 45, 257-273. 31. Vali, Z., and Pathy, L. (1982) J. Biol. Chem. 257, 2104-2110. 32. Levin, E. G., Marzec, U., Anderson, J., and Harker, L. A. (1984) J. Clin. Invest. 74, 1988-l 995. 33. Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412. 34. Miles, L. A., Dahlberg, C,. M., Plescia, J., Felez, J., Kato, K., and Plow, E. F. (1991) Biochemistry 30, 1682-1691. 35. Harpel, P. C., Gordon, B. R., and Parker, T. S. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3847-3851. 36. Loscalzo, J., Weinfeld, M., Fless, G. M., and Scanu, A. M. (1990) Atherosclerosis 10, 240-245. 37. Edelberg, J. M., Gonzalez-Gronow, M, and Pizzo, S. V. (1990) Thromb. Res. 57, 155-162. 38. Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919. 39. Fears, R., Hibbs, M. J., and Smith, R. A. G. (1985) Biochem. J. 229, 555558. 40. Rijken, D. C., Hoylaerts, M., and Collen, D. (1982) J. Biol. Chem. 257, 2920-2925. 41. Zamarron, C., Lijnen, H. R., and Collen, D. (1984) J. Biol. Chem. 259, 2080-2083.
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THE BINDING OF PLASMINOGEN FRAGMENTS TO CULTURED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS Hua-Lin Wu, Ing-Shiang Wu, Ro-Yun Fang, Jen-Shau Hau, Dung-Ho Bi-lng Chang#, Tsun-Mei Lit-@, and Guey-Yueh Shi*
Wu,
Departments of Biochemistry and #Medical Technology, Medical College, National Cheng Kung University, Tainan, Taiwan 700, Republic of China Received
August
25,
1992
Glu-plasminogen, kringle l-5, kringle l-3; and miniplasminogen exhibited strong binding to human umbilical vein endothelial cells (HUVEC). On the other hand, no significant binding was obtained with microplasminogen and kringle 4. Kringle l-5 and miniplasminogen, which both contained kringle 5, specifically inhibited the binding of plasminogen to HUVEC while kringle l-3 did not. The results implied plasminogen molecule contained at least two binding sites, with which it interacted HUVEC. The stronger binding site was located in kringle 5 and the weaker one was in kringle 1-3. Kringle 4 and the active site domain exhibited no significant binding to HUVEC. The interaction of plasminogen with HUVEC 0 1992Academic Press,Inc. is mainly through binding site on kringle 5.
Vascular endothelial cells play an important role in the regulation of the human fibrinolytic system (1). Plasminogen activators and plasminogen activator-inhibitor are synthesized and secreted by endothelial cells (2-6). Furthermore, endothelial cells provide binding sites for plasminogen and tissue-type plasminogen activator (7-10). The binding of plasminogen to endothelial cell surface enhances its activation by tissue-type plasminogen activator (11). The interaction of plasmin with endothelial cells might also be important in the feed back regulation *To whom correspondence should be addressed. The abbreviations used are: Glu-Pig, native plasminogen consisting of Glul-Asn7so; Mini-Plg, Micro-Plg, kringle 1-5, kringle l-3, and kringle 4 corresponding to plasminogen fragments consisting of Va1442-Asn7sop Lysssc-Asn790, Lys77-Arg529, Lys77-Valss7, and Vals54-Ala439, respectively; SDS, sodium dodecyl sulfate; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline; BSA, bovine serum albumin. 0006-291X/92
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of fibrinolytic activity of the cells (12). It has been demonstrated that lipoprotein(a) binds to the plasminogen binding site on endothelial cells (13, 14) and may thereby inhibit plasminogen activation by competing for the same binding site. Clinical studies also demonstrate that patients with elevated lipoprotein(a) levels have less fibrinolytic activity (15). Lipoprotein(a) contains a lipid core, an apoprotein B and an apoprotein(a) subunits (16). Protein and cDNA sequence analysis indicate a high degree of homology between apoprotein(a) and plasminogen (17). The apoprotein(a) subunit contains up to 37 copies of kringle 4 and a single copy of kringle 5, with 75-85% and 95% homology, respectively, to their plasminogen counterparts (17). Lipoprotein(a) inhibited the binding of plasminogen form 2 to monocytoid U937 cells as well as to HUVEC (14). In addition, inhibition of [1251]-plasminogen form 2 to U937 cells was blocked by monoclonal antibodies against kringle 4 (18). Thus, kringle 4 was suggested to be responsible for the binding of lipoprotein(a) to U937 Miles et al reported that lipoprotein(a) inhibited the binding of cells. [12sl]-labeled plasminogen at the same extent as that of unlabeled plasminogen (14). However, isolated kringle 4 was 100 to 300 times less potent an inhibitor of plasminogen binding to U937 and to HUVEC than lipoprotein(a) although it has similar conformation, immunochemical and functional properties as integrated kringle 4 (14). Therefore, it is suggested that the capacity of lipoprotein(a) to compete with plasminogen for cellular binding must reside in concatenation of kringle 4-like structures with lipoprotein(a). The binding can also be attributable to other homologies between the molecules such as the kringle 5 and the light-chain domain of lipoprotein(a) which is 94% homologous to the plasminogen proteinase domain (14, 19). The result obtained using platelets as models demonstrates that kringle l-3 is a primary recognition site for plasminogen binding to both thrombin-stimulated and non-stimulated platelets (20). In addition, kringle 4 and Mini-Plg, which contains kringle 5 and proteinase domains, can also participate in the interaction (20). Therefore, it is still uncertain which domain of either lipoprotein(a) or plasminogen is responsible for their binding to HUVEC. Plasminogen fragments of different sizes have been isolated by partial digestion of plasminogen with elastase and plasmin to form Mini-Plg, kringle l-3, kringle 4, Micro-Plg, and kringle 1-5 (21-25). Human plasminogen and its fragments were applied to study the interaction of plasminogen with fibrin (25). The results indicated that there are two fibrin-binding domains in plasminogen; the one in kringle 5 is of higher affinity and the other in kringle 1-3 is of lower affinity (25). The present study was designed to investigate which domain of plasminogen was 704
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Methods
Preoaration of olasminocen and olasmin: Human plasminogen was prepared from pooled human plasma by a modification of the Deutsch and Mertz method (26). Forms 1 and 2 of native plasminogen were separated by chromatography on Lys-Sepharose (27). For this study, form 2 of human plasminogen was used exclusively. Urokinase-free human plasmin was prepared by activation of human plasminogen with urokinase-Sepharose as described in a previous report (22). Preoaration of Mini-Plo. Micro-Plc. krinole 1-5. krinale l-3. a d mole 4: Mini-Plg, kringle 1-3, and kringle 4 were obtained by dige?sting Glu-Plg with a catalytic amount of porcine pancreatic elastase and purified by Sephadex G-75 (2.5 x 70 cm) and Lys-Sepharose (2 x 15 cm) columns (20, 24, 27). Micro-Plg and kringle l-5 were made by incubating Glu-Pig with plasmin in an alkaline solution and purified by Lys- and soybean-trypsin-inhibitor-Sepharoses as in the previous reports (24, 25, 28). Protein concentration: The protein spectrophotometrically using the following molecular weights, respectively: Glu-Pig, Plg, 16.0 and 38,000 (21); Micro-Plg, 16.0 17.0 and 55,000 (25). The E 28onm values 4 were 8.08 x 104 M-‘cm-1 and 3.1 x 104
concentrations were determined &I%, 280 nm values and 17.0 and 92,000 (29, 30); Miniand 28, 617 (24); kringle l-5, used for kringle l-3 and kringle M-‘cm-1, respectively (31).
[ZI$ labeling of protein: 100 ul of protein sample at a concentration of 1 .O mg/ml was incubated with Nat251 (100 uCi) and lightly agitated at room temperature in an Eppendorf tube previously coated with 100 1.11of lodo-gen (0.08 mg/ml) for 8 min. After incubation, the reaction mixture was applied to a PD-10 column and eluted with 0.01 M phosphate buffer, pH 7.2. The labeled protein was collected after The specific radioactivities varied from 2 x discarding the void volume. 10s cpm to 5 x 10s cpm/mg of protein. Cell culture: Endothelial cells were isolated from human umbilical cord veins by reaction with collagenase following the previously described method (32). Cells were grown to confluence in medium M-199 containing 10% fetal calf serum, 25 ug/ml endothelial cell growth factor, 10 units/ml heparin, 100 units/ml penicillin, and 100 units/ml Passaged cells were subcultured in 96-well dishes and streptomycin. allowed to grow for 3 days to confluence under the same condition as Third passaged cultures were employed in all primary cultures. experiments. The cell count at confluency averaged 3 x 104 per well. Prior to using live cells in binding studies, the confluent cell culture was washed 5 times with 0.1 M PBS, pH 7.4 containing 0.25% BSA. 705
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hvde-fixed c&: The HUVEC monolayer was washed 5 times with M-199, once in 0.1 M PBS, pH 7.4 at 37OC and then fixed in 0.02% (v/v) glutaraldehyde in PBS at 37X for 20 min as described (11). Fixed cells were washed 3 times with PBS-Tween 20-BSA (0.1 M PBS, pH 7.4 containing 0.5% Tween 20 and 0.1% BSA) and passivated 60 min in the same buffer. Binding studies were performed after wash fixed cell monolayers 3 times with 0.1 M PBS, pH 7.4 containing 0.25% BSA. Bindina of olasminoaen fraaments to HUVEC: Measurement of binding of [1251]-Giu-Pig to HUVEC was carried out using live and fixed endothelial cell monolayers. The binding of plasminogen fragments such as Mini-Plg, Micro-Plg, kringle l-5, kringle 1-3, or kringle 4 to HUVEC was done using fixed cells. [125l]-labeled protein at various concentrations in 100 ~1 PBS containing 0.25% BSA and 0.1 p.M aprotinin, with or without 10 mM tranexamic acid were added to triplicate tissue culture wells containing live or fixed cell monolayers. The tissue culture plates were incubated at 4OC for 30 min and 50-pl aliquots of the culture supernatant were removed from the wells and counted in an LKB y-counter (value A). The wells were then washed 5 times with PBS (total wash time 45 s) and the cells in each well were solubilized in 150 p.1 of 1% SDS, 0.5 M NaOH and 0.01 M EDTA (11). The radioactivities of total cell lysates were measured with a y-counter (value B). The amount of protein bound to HUVEC per well = the amount of protein added x B /(2A + B). Specific binding was calculated as binding in the absence of tranexamic acid (total binding) minus binding in the presence of tranexamic acid (nonspecific binding). The molecules of ligand bound per cell were calculated from the specific binding of each protein. Cell number was determined by trypsinization of the cells from wells on each 96-well tissue culture plates and by counting the cells under a microscope. SDS oolvacrvlamide ael electroohoresis: The basic techniques electrophoresis was done as described by Weber and Osborn (33).
of gel
Reagents: lodo-gen, BSA. tranexamic acid and elastase were purchased from Sigma. Sepharose 48 and PD-10 were from Pharmacia LKB Biotechnology. Endothelial cell growth factor was from Collaborative Incorporated. Medium M-199, fetal calf serum, penicillin and streptomycin were from Gibco. All other reagents used were analytical grade. Results The binding of human Glu-Pig to HUVEC was a concentration-dependent reaction. Pretreatment of HUVEC with glutaraldehyde did not significantly alter the binding of Glu-Pig to the cells (Fig. 1). The endothelial cells pretreated with glutaraldehyde were used for binding assays, since less cell detachment was observed and more consistent 706
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Fig 1. Binding of [tW]-Glu-Plg to HUVEC. Fixed and living HUVEC monolayers were incubated with [ 12sl]-Glu-Pig for 30 min at 4% in the presence or absence of 10 mM tranexamic acid. The total binding ( o), nonspecific binding ( l ), specific binding (m) of fixed cells, and the specific binding (A) of living cells were determined as described under “Materials and Methods”. Each data point represents the mean of triplicate well experiments. Fig. 2. Effect of unlabeled Glu-Pig on the binding of [12sl]-Glu-Pig to HUVEC. Fixed HUVEC were incubated with 0.1 to 1 uM of labeled Glu-Pig and 0.9 to 0 PM unlabeled Glu-Pig at a final total concentration of I PM for 30 min at 4% in the presence or absence of 10 mM tranexamic acid. Specific binding was determined by subtracting “nonspecific” (nontranexamic acid inhibitable) binding from total binding at each incubation. Each data point represents the mean f S.D. of triplicate samples.
results could be obtained. The binding of Glu-Plg to HUVEC was specifically inhibited by 10 mM tranexamic acid (Fig. 1). Therefore, tranexamic acid was added to the samples to obtain the values of Specific binding was calculated by subtracting nonspecific binding. “nonspecific” (non-tranexamic acid inhibitable) binding from total binding Native unlabeled Glu-Plg and [1251]-Glu-Plg at each ligand concentration. had the same efficiency in the competition for the binding to HUVEC (Fig. 2). Therefore, radioiodination of Glu-Plg did not alter the binding property. The number of molecules of ligand bound per cell at a added were approximately 2 x 107 per concentration of 1 uM [ 125l]-Glu-Pig fragments, which contained only cell (Fig. 1 and 2). Purified plasminogen one major protein band in each fraction as analyzed by SDS-gel The molecular weights of Mini-Plg, electrophoresis, were prepared. Micro-Plg, kringle 4, kringle l-3, and kringle 1-5 were 38, 28, 10, 33, and 55 KDa, respectively (Fig. 3). Mini-Plg, kringle l-5, and kringle 1-3 bound 707
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KDa -94 -07
abcdef
Fig. 3. SDS polyacrylamide gels of plasminogen fragments. Lane a:MiniPlg; b:Micro-Plg; c:Kringle 4; d:Kringle 1-3; e:Kringle 1-5; f:Glu-Plg.
to HUVEC dose-dependently (Fig. 4). However, Micro-Plg and kringle 4 did not have significant binding to HUVEC. The binding potency of plasminogen fragments to HUVEC was in the order of Glu-Pig > Mini-Plg > kringle I-5 > kringle 1-3 (Fig. 4). The ability of plasminogen fragments at various concentrations to compete with [ 1251]-Glu-Pig (1 PM) for binding to HUVEC
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Fig. 4. Binding of plasminogen fragments to HUVEC. [t2sl]-Glu-Pig ( l ), [12sl]-Mini-Plg (AL), [12sl]-kringle l-5 (o), [12sl]-kringle 1-3 ( n), [‘=I]kringle 4 ( q), or [12sl]-Micro-Plg (A) was added at increasing concentrations in the presence or absence of 10 mM tranexamic acid to determine specific binding as described in the legend to figure 2. Each data point represents the mean f S.D. of triplicate samples. Fig. 5. Inhibition of [12sl]-Glu-Plg binding to HUVEC with excess unlabeled plasminogen fragment. [lW]-Glu-Plg (1 PM) and unlabeled Glu-Plg ( l ), kringle 1-5 ( o), Mini-Plg (A), or kringle l-3 ( n) were incubated with HUVEC for 30 min at 4%. The specific binding of each incubation was determined as in the legend to figure 2. Each data point represents the mean f S. D. of triplicate samples. 708
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was studied. The binding of [ 1251]-Glu-Pig to endothelial cells was inhibited by kringle 1-5 and Mini-Plg as well as by unlabeled Glu-Pig, but not by kringle 1-3 (Fig. 5). The intact Glu-Pig was a more effective competing ligand than any of the plasminogen fragments, and the kringle 5 containing plasminogen fragments, kringle 1-5 and Mini-Plg, were also effective in inhibiting the binding of [125l]-Glu-Plg to HUVEC. Discussion Plasminogen was shown to bind to a variety of cell types including platelets, monocytes, lymphocytes, endothelial cells, smooth muscle cells, and fibroblasts (8, 20). Recent study of Miles et al indicated that a-enolase was a candidate plasminogen receptor on U937 cells (34). This study demonstrated that plasminogen specifically bound to HUVEC, and the number of Glu-Pig molecules bound per cell were 2 x 107 for HUVEC at an input Glu-Pig concentration of 1 PM. This result is comparable with those obtained in previous studies (7, 8). The results of this study indicate that the binding of plasminogen to HUVEC was mainly through kringle 5, since kringle 5containing plasminogen fragments, kringle l-5 and Mini-Plg, can effectively compete for the binding of [ f251]-Glu-Pig to HUVEC (Fig. 5). On the other hand, kringle l-3 did not inhibit the binding of [1251]-Glu-Pig to HUVEC (Fig. 5). Thus, kringle 5 should exhibit the strong binding site and kringle l-3 the weak binding site. The similar results were obtained in the studies of plasminogen binding to fibrin (25). Several studies indicated that lipoprotein(a) may modulate fibrinolysis by competing with plasminogen for binding sites on both fibrin and fibrinogen (35-37). Since binding of lipoprotein(a) to fibrin and fibrinogen increases with partial proteolysis with plasmin, lysine-binding sites are assumed to be involved in the binding of lipoprotein(a) to fibrin and fibrinogen (35). Therefore, kringle 4 and perhaps kringle 5 domains of lipoprotein(a) are suggested to be responsible for the binding of lipoprotein(a) to fibrin and fibrinogen. Results from studies using U937 monocytoid and endothelial cells demonstrate that lipoprotein(a) binds to these cells with an affinity equal to that of plasminogen (13, 18). This observation is to some extent unexpected since lipoprotein(a) has such a higher number of potential binding sites in its many repeated kringles as compared to plasminogen which contains only one copy of both kringle 4 and kringle 5. It is therefore suggested that only a few of kringle 4 present in lipoprotein(a) may be available for binding with cells, or other kringle such as kringle 5 may be responsible for the interaction. This study indicates that kringle 5 of plasminogen, not kringle 4, is responsible 709
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for the interaction of plasminogen with HUVEC. This observation is consistent with the result that krjngle 4 is less effective in inhibition of plasminogen binding to HUVEC (14). The binding of plasminogen to either fibrin or endothelial cell surface enhances its activation by plasminogen activators (11, 38-41) is also consistent with the observation that binding of plasminogen to both fibrin and endothelial cells is mainly determined by kringle 5. Whether lipoprotein(a) interacts with endothelial cells through kringle 4 or 5 is subjected to further studies. Acknowledamen&: The expert technical assistance Tsai and S. -F. Chen are gratefully acknowledged. supported by Grant NSC 80-0412-B008-80R, NSC NSC 81-0412-B006-18 from the National Science China.
of L. -C. Chang, C. -F. This work was 80-0412-B006-06, and Council, Republic of
References 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11 12.
13. 14. 15. 16.
Levin, E. G., and Loskutoff, D. J. (1982) Ann. N. Y. Acad. Sci. 401, 184194. Bachmann, F., and Kruithof, E. K. 0. (1984) Semin. Thromb. Haemost.. 10, 6-17. Van Mourik, J. A., Lawrence, D. A., and Loskutoff, D. J. (1984) J. Biol. Chem. 259, 14914-14921. Kruithof, E. K. O., Tran-Thang, C., Ransijn, A., and Bachmann, F. W. (1984) Blood 64, 907-913. Loskutoff, D. J., van Mourik, J. A., Erickson, L. A., and Lawrence, D. (1983) Proc. Natl. Sci. U.S.A. 80,6804-6808. Ramakrishnan, V., Sinicropi, D. V., Dere, R., Darbonne, W. C., Bechtol, K. B., and Baker, J. B. (1990) J. Biol. Chem. 265, 2755-2762. Miles. L. A., Levin, E. G., Plescia, J. Collen, D., and Plow, E. F. (1988) Blood 72, 628-635. Miles, L. A., and Plow, E. F. (1988) Fibrinolysis 2, 61-71. Hajjar, K., and Hamel, N. M. (1990) J. Biol. Chem. 265, 2908-2916. Russell, M. E., Quertermous, T., Declerck, P. J., Collen, D., Haber, E., and Homey, C. J. (1990) J. Biol. Chem. 265, 2569-2575. Hajjar, K. A., Harpel, E. A., Jaffe, E. A., and Nachman, R. L. (1986) J. Biol. Chem. 261, 11656-l 1662. Shi, G. -Y., Hau, J. -S., Wang, S. -J., Wu, I. -S., Chang, B. -I., Lin, M. T., Chow, Y. -H., Chang, W. -C., Wing, L. -Y. C., Jen, C. J., and Wu, H. -L. (1992) J. Biol. Chem. (in press). Hajjar, K. A., Gavish, D., Breslow, J. L., and Nachman, R. L. (1989) Nature 339, 303-305. Miles, L. A., Fless, G. M., Levin, E. G., Scanu, A. M., and Plow, E. F. (1989) Nature 339, 301-303. Hamsten, A., Walldius, G., Szamosi, A. Blomback, M., De Faire, U., Dahlen, G., Landou, C., and Wiman, B. (1987) Lancet ii, 3-9. Scanu, A. M. (1988) Arch. Pathol. Lab. Medicine 112, 1045-1047. 710
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17. McLean, J. W., Tomlinson, J. E., Kuang, W. -J., Eaton, D. L., Chen, E. Y., Fless, G. M., Scanu, A. M., and Lawn, R. M. (1987) Nature 330, 132137. 18. Gonzales-Gronow, M., Edelberg, J. M., and Pizza, S. V. (1989) Biochemistry 28, 2375-2379. 19. Edelberg, J. M., and Pizzo, S. V. (1991) Fibrinolysis 5, 135-143. 20. Miles, L. A., Dahlberg, C. M., and Plow, E. F. (1988) J. Biol. Chem. 263, 11928-l 1934. 21. Sottrup-Jensen, L., Claeys, H., Zajdel, M., Petersen, T. E., and Magnusson, S. (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191209. 22. Wu, H. -L., Shi, G. -Y., and Bender, M. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8292-8295. 23. Wu, H. -L., Shi, G. -Y., Wohl, R. C., and Bender, M. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8793-8795. 24. Shi, G. -Y., and Wu, H. -L. (1988) J. Biol. Chem. 263, 17071-17075. 25. Wu, H. -L., Chang, B. -I., Wu, D. -H., Chang, L. -C., Gong, C. -C., Lou, K. L., and Shi, G. -Y. (1990) J. Biol. Chem. 265, 19658-19664. 26. Deutsch, D. G., and Mertz, E. T. (1970) Science 170, 1095-1096. 27. Brockway, W. J., and Castellino, F. J. (1972) Arch. Biochem. Biophys. 151, 194-199. 28. Shi, G. -Y., Wu, D. -H., and Wu, H. -L. (1991) Biochem. Biophys. Res. Commun. 178, 360-368. 29. Violand, B. N., and Castellino, F. J. (1976) J. Biol. Chem. 251, 3906391 2. 30. Robbins, K. C., and Summaria, L. (1976) Methods Enzymol. 45, 257-273. 31. Vali, Z., and Pathy, L. (1982) J. Biol. Chem. 257, 2104-2110. 32. Levin, E. G., Marzec, U., Anderson, J., and Harker, L. A. (1984) J. Clin. Invest. 74, 1988-l 995. 33. Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412. 34. Miles, L. A., Dahlberg, C,. M., Plescia, J., Felez, J., Kato, K., and Plow, E. F. (1991) Biochemistry 30, 1682-1691. 35. Harpel, P. C., Gordon, B. R., and Parker, T. S. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3847-3851. 36. Loscalzo, J., Weinfeld, M., Fless, G. M., and Scanu, A. M. (1990) Atherosclerosis 10, 240-245. 37. Edelberg, J. M., Gonzalez-Gronow, M, and Pizzo, S. V. (1990) Thromb. Res. 57, 155-162. 38. Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919. 39. Fears, R., Hibbs, M. J., and Smith, R. A. G. (1985) Biochem. J. 229, 555558. 40. Rijken, D. C., Hoylaerts, M., and Collen, D. (1982) J. Biol. Chem. 257, 2920-2925. 41. Zamarron, C., Lijnen, H. R., and Collen, D. (1984) J. Biol. Chem. 259, 2080-2083.
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