SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 3, 1992

Effect of Human α-Thrombin on the Transforming Growth Factor-ß1 -Binding Activity of Human α2-Macroglobulin

Thrombin plays a central role in hemostasis by func­ tioning in coagulation and platelet activation.1In addi­ tion, α-thrombin mediates numerous biologic responses, including wound repair, inflammation, and bone resorp­ tion.1-3 Many of these activities result from α-thrombin's association with specific cellular receptors. A thrombin receptor mediating cellular growth and platelet activation was recently cloned and sequenced.4 The structure of the thrombin receptor includes a substrate site for the prote­ olytic activity of α-thrombin and a noncovalent binding site that interacts with the α-thrombin anion exosite.4,5 Some growth regulatory activities of proteinases may reflect interaction with cytokines. For example, the latent form of transforming growth factor-ß1 (TGF-ß1) may be activated by plasmin.6 The 25 kd active form of TGF-ß1 expresses proliferative and anti-proliferative activity. TGF-ß1 is a major constituent of platelet α-granules, released on platelet activation, and active in many of the same biologic processes as α-thrombin (wound repair, inflammation, bone remodeling).7 The activity of TGF-ß1 depends on association with specific cellular TGF-ß receptors.7 Therefore any macromolecule that binds TGF-ß1 and alters the availability of this cytokine to cellular TGF-ß receptors might alter the apparent activity of TGF-ß1 toward cells. α2-Macroglobulin (α2M) is a high molecular weight proteinase inhib­ itor (Mr 718,000) and a TGF-ßl-binding protein.8"13 When α2M reacts with proteinases, the inhibitor under­

From the Departments of Pathology and Biochemistry, University of Virginia Health Sciences Center, Charlottesville, Virginia. Reprint requests: Dr. Gonias, Associate Professor of Pathology and Biochemistry, University of Virginia Health Sci. Cntr., Depart­ ment of Pathology, Box 214, Charlottesville, VA 22908.

goes a major conformational change.8 A comparable conformational change may be induced by direct aminolysis of the α2M thiolester bonds by methylamine.14 The conformation of α2M after reaction with proteinase or methylamine has been referred to as the fast-form (based on mobility in native polyacrylamide gel electrophoresis systems).15 α2M fast-forms are recognized by specific cellular receptors; native α2M demonstrates no affinity for the receptor.16 The α2M receptor has been purified and is identical to low density lipoprotein (LDL) recep­ tor-related protein (LRP). 17,18 The TGF-ß1-binding activity of α2M is signifi­ cantly increased after a 2 M undergoes conformational change. 10-13,19 This is particularly important, since α2Mproteinase complexes and α2M-methylamine may deliver TGF-ß1 to the cell surface via a direct interaction with LRP.13 Previous studies have shown that TGF-ß1 binds to α2M-plasmin,11 α2M-trypsin, α2M-elastase, and α2M-chymotrypsin.19 The latter three complexes may be formed so that either 1 mol of proteinase is bound per mol of α2M (binary complex) or 2 mol of proteinase are bound per mol of α2M (ternary complex). α-Thrombin reacts with α2M to form almost exclusively binary α2Mthrombin complex.20'21 The resulting α2M-thrombin preparations include a high percentage of relatively stable α2M conformational intermediates (structures that have undergone partial or incomplete structural transforma­ tion). The effects of α-thrombin on the interaction of α2M with TGF-ß1 have not been completely evaluated. In this investigation, we demonstrate that α2M-thrombin binds increased levels of TGF-ß1 compared with native α2M. Our animal model experiments show that TGF-ß1 α2M-thrombin complex binds to LRP and that this inter­ action is responsible for the plasma clearance of TGF-ß1 associated with α2M.

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DONNA J. WEBB, B.S., JONATHAN LaMARRE, D.V.M., Ph.D., and STEVEN L GONIAS, M.D., Ph.D.

SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 3, 1992

MATERIALS AND METHODS Proteins and Reagents Human-α thrombin was a generous gift from Dr. John Fenton II (Albany, NY). Trypsin, p-nitrophenyl p'-guanidinobenzoate HC1 (PNPGB), and methylamineHC1 were purchased from Sigma (St. Louis, MO). The concentration of active trypsin was determined by the method of Chase and Shaw.22 D-Phe-Pro-ArgCH2Cl (PPACK) was purchased from Calbiochem (San Diego, CA). α2M was purified according to the procedure of Imber and Pizzo.23 α2M-methylamine was prepared by dialyzing native α2M against 200 mM methylamine, pH 8.2 for 6 hours at 22°C followed by extensive dialysis against 20 mM sodium phosphate, 150 mM sodium chlo­ ride pH 7.4 (phosphate buffered saline [PBS]). TGF-ßl was purified from acid/ethanol extracts of human platelets according to the procedure of Assoian.24 TGF-ß1 was radioiodinated according to the procedure of Ruff and Rizzino.25 The specific activity of 125I-TGF-ß1 ranged from 50 to 100 |xCi/|xg.

Formation of Transforming Growth Factor-ß1-α2-Macroglobulin-Proteinase Complexes α2M-proteinase complexes were prepared by react­ ing native α2M (0.5 µM) with trypsin (1.0 |xM) or thrombin (0.45 to 4.5 µM) in PBS for 30 minutes at 22°C, unless otherwise specified. The α2M-associated proteinase was inactivated with 0.4 mM PNPGB (trypsin) or 0.2 mM PPACK (α-thrombin).

Electrophoresis Native α2M, α2M-proteinase and α2M-methylamine (0.4 |xM) were incubated with 125I-TGF-ß1 (2.5 nM) for 1.5 hours at 37°C. The solutions were subjected to native polyacrylamide gel electrophoresis (PAGE) on 5% slabs, as previously described.13 Binding of 125 ITGF-ß1 to the various forms of α2M was detected by autoradiography and quantified by slicing each lane into 3 mm sections. The radioactivity in the sections was determined in a gamma counter.

Plasma Clearance Experiments α2M-thrombin was prepared by incubating 0.8 µM α2M with 1.5 (µM α-thrombin for 30 minutes at 22°C. PPACK (0.2 mM) was then added to inactivate the α-thrombin. 125I-TGF-ß1 (2.6 nM) was incubated with

α2M-thrombin or α2M-methylamine (0.7 µM) for 1.5 hours at 37°C. Complexes of 125I-TGF-µ1 with α2Mmethylamine or α2M-thrombin were purified by chroma­ tography on Superose-6 (flow rate, 0.4 ml/min). Plasma clearance studies were performed using 20-week old CD-1 female mice (Charles River Breeding Laboratories, Wilmington, MA) as previously described.13 The puri­ fied 125I-TGF-ß1-α2M complexes (400 (µ1) were injected into the lateral tail veins of anesthetized mice. Beginning at 10 seconds, blood samples (25 |xl) were taken from the retro-orbital venous plexus using heparinized hematocrit tubes. The radioactivity in each blood sample was deter­ mined in a gamma counter and plotted as a percentage of the radioactivity in the 10-second sample. Clearance competition experiments were performed by coinjecting purified 125I-TGF-ß1-α2M-thrombin and 1.0 mg of α2Mmethylamine.

Organ Distribution Studies Purified 125I-TGF-ß1-α2M-methylamine and 125 ITGF-ß1-α2M-thrombin were injected intravenously into mice. After 45 minutes, the animals were sacrificed. The major organs were recovered intact, rinsed in normal saline, blotted, and weighed. The radioactivity in each organ was determined in a gamma counter and expressed as a percentage of the total recovered radioactivity. These values were then normalized for organ mass by dividing the percentage of recovered radioactivity by the weight of the organ in grams.

RESULTS AND DISCUSSION The initial reports suggesting preferential binding of TGF-ß1 to α2M fast form were based on comparisons of native α2M and α2M-methylamine.10,13 For α2M-proteinase complexes, the factors determining TGF-ß1 binding activity are more complex. Recent studies from this laboratory have demonstrated that many α2M-proteinase complexes, including α2M-trypsin, express en­ hanced TGF-ß1-binding activity compared with native α2M when one of the two proteinase binding sites is occupied.19 Saturation of the second proteinase binding site to form ternary α2M-proteinase complex (2 mol pro­ teinase per mol α2M) results in a significant decrease in TGF-ß1 binding activity. α2M-thrombin complex was prepared by reacting 0.5 µM α2M with 0.45 to 4.5 µM active α-thrombin. By native PAGE, the resulting preparations demonstrated increased mobility compared with native α2M; however, the majority of the protein still migrated less rapidly than ternary α2M-trypsin or α2M-methylamine (Fig. 1). As demonstrated by autoradiography, 125I-TGF-ß1 bound to α2M-thrombin. The level of binding was higher

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than that demonstrated with native α2M (272 µmol 125ITGF-ß1/mol α2M-thrombin versus 137 (µmol 125I-TGFpi/mol native α2M), but lower than that demonstrated with α2M-methylamine. In this respect, α2M-thrombin complex was similar to binary α2M-trypsin and binary α2M-chymotrypsin studied elsewhere. 19 Unlike the latter two complexes, binary α2M-thrombin does not readily bind additional α-thrombin to form ternary complex as the concentration of α-thrombin is increased.20'21 This probably explains why the TGF-ß1-binding activity of α2M-thrombin was not decreased when the complex was prepared by incubating native α2M with up to a ninefold molar excess of α-thrombin. For comparison, Figure 1 shows that ternary α2M-trypsin (formed by incubating

α2M with a twofold molar excess of active trypsin) binds decreased levels of 125I-TGF-ß1. By autoradiography, the mobility of the l25 ITGF-ß1 bound to native α2M was actually somewhat faster than the mobility of the Coomassie-stained slow form band (lane a), as previously demonstrated.1319 Trace contamination of our native α2M preparation with molecules that have undergone partial conformational change may be responsible. It has been demonstrated previously that trypsin re­ acts with α2M-thrombin, causing further conformational change in the α 2 M. 20,21 Stable heterocomplexes of α2M with thrombin and trypsin are formed. As shown in Fig­ ure 2, trypsin-treated α2M-thrombin migrated equiva-

FIG. 2. Native polyacrylamide gel electrophoresis (PAGE) and autoradiography of 125l-transforming growth factor (TGF)-ß1 binding to α2-macroglobulin (α2M)-thrombin reacted with trypsin. α2M-thrombin was prepared by incubating native α2M (0.64 (µM) with α-thrombin (0.6 or 1.2 µM)for30 minutes at 22°C. The α2M-thrombin preparations were then incubated with a onefold or twofold molar excess of trypsin for 30 minutes at 22°C. PPACK 0.2 mM and PNPGB 0.4 mM were added to each solution. 125 l-TGF-ß1 binding was studied by native PAGE and autoradiography. Lane a shows native α2M. Lane b shows α2M-thrombin formed with a slight excess of α2M (α2M-thrombin preparation I). Lane c shows α2M-thrombin formed with a 1.9-fold molar excess of α-thrombin (preparation II). Lane d shows preparation I incubated with an equimolar concentration of trypsin. Lane e shows preparation I incubated with a twofold molar excess of trypsin. Lanes f and g show preparation II incubated with an equimolar concentration or a twofold molar excess of trypsin, respectively. Lane h shows α2M-methylamine.

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FIG. 1. Native polyacrylamide gel electrophoresis (PAGE) and autoradiography of 12S l-transforming growth factor (TGF)-ß1 binding to α2-macroglobulin (α2M). 125ITGF-ß1 (2.5 nM) was incubated with native α2M, α2M-thrombin, α2M-trypsin, or α2Mmethylamine (0.4 µM). The samples were then subjected to native PAGE and autora­ diography. Native α2M is shown in lane a. α2M-thrombin formed at molar ratios of 1:0.9, 1:1.8, 1:4.5, and 1:9.0 (α2M:α-throm­ bin) are shown in lanes b, c, d, and e, re­ spectively. α2M-trypsin formed by incubat­ ing native α2M with a twofold molar excess of trypsin is shown in lane f. α2M-methylamine is shown in lane g.

SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 3, 1992

FIG. 3. Plasma clearance of 125l-transforming growth factor (TGF)-ß1-α2-macroglobulin (α2M) complexes. The plasma clearance profiles of 125l-TGF-ß1-α2M-methylamine (•), 125l-TGF-ß1-α2M-thrombin (■), and 125l-TGF-ß1-α2M-thrombin coinjected with 1 mg of α2M-methylamine (A) are shown. The 125l-TGF-ß1-α2M preparations were purified by chromatography on Superose-6 imme­ diately prior to injection.

lently to ternary α2M-trypsin in the native PAGE system. The α2M-thrombin/trypsin demonstrated decreased 125 ITGF-ß1 binding activity. This result most likely reflects occupancy of the second proteinase binding site by trypsin. 125 I-TGF-ßl-α2M-methylamine and 125 I-TGF-ßlα2M-thrombin were purified by chromatography on Su­ perose-6. The plasma clearance of each complex was studied in mice (Fig. 3). Clearance of 125I-TGF-ß1-α2Mmethylamine was an apparent first-order process. The plasma half-life was 3 to 5 minutes, confirming previ­ ously reported results.13 Clearance of 125I-TGF-ß1 bound to α2M-methylamine is mediated by LRP. 13 The clearance of TGF-ß1 bound to an α2M-proteinase com­ plex has not been studied. Figure 3 shows that the plasma clearance of 125 ITGF-ß1-α2M-thrombin was similar but not identical to the clearance of 125I-TGF-ß1-α2M-methylamine. Ap­ proximately 50% of the radioligand was eliminated from the plasma within 5 minutes; however, the clearance profile was nonlinear and 15% of the radioligand re­ mained in the circulation at 15 minutes. The plasma clearance of 125I-TGF-ß1-α2M-thrombin was equivalent to the clearance of α2M-thrombin (prepared with 125 Iα2M) reported elsewhere.21 To demonstrate that LRP is responsible for the clearance of TGF-(ß1 bound to α2M-thrombin, purified 125 I-TGF-ß1-α2M-thrombin was coinjected with a large

molar excess of nonradiolabeled α2M-methylamine. The function of the nonradiolabeled ligand is to saturate the α2M-receptor in vivo and prolong radioligand clearance dependent on this receptor.16 As shown in Figure 3, the clearance of 125I-TGF-ß1-α2M-thrombin was markedly inhibited by α2M-methylamine, confirming the role of LRP in this process. Table 1 presents the organ distribution of 125I-TGFß1-α2M-thrombin. The recovery of radioactivity primar­ ily in the liver (even after normalizing results for organ mass) is consistent with the role of LRP in the clearance of this complex. By contrast, 125I-TGF-ß1 injected alone is recovered in the lungs, kidneys, and liver after intravascular injection.13,26

CONCLUSIONS The reaction of α-thrombin with human α2M, under conditions specified here, results in the formation of α2M-thrombin complexes in which the α2M has under­ gone partial or incomplete conformational change. The studies presented in this investigation demonstrate that α2M-thrombin binds TGF-ß1; the level of binding detected by native PAGE is increased compared with native α2M. α2M-thrombin, like other α2M fast forms, is cleared from the plasma by LRP located primarily in the liver.

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TABLE 1. Organ Distribution of α2M-TGF-ß1 Complexes TGF-ß1-α2M-thrombin

Organ

(%)

Normalized Radioactivity (%/gm)

Liver

Radioactivity Recovered

(%)

Normalized Radioactivity (%/gm) 69

94

57

95

Lungs

1

5

1

2

Kidneys

3

4

2

4

Heart

1

2

1

1

Spleen

2

15

2

14

The α2M-fast form-LRP interaction is not inhibited by TGF-ß1 that is bound to α2M. As a result, fast forms of α2M like α2M-thrombin may deliver TGF-01 to the sur­ face of cells that express LRP. Based on the in vivo experiments presented here, the fate of TGF-ß1 after delivery to the cell as a complex with α2M cannot be determined. The factors determining affinity of TGF-ß1 for α2M are complex; however, the conformation of α2M is ap­ parently important. We believe that during α2M confor­ mational change, a critical TGF-01 binding site is ex­ posed. Proteinases that bind to α2M and form ternary complexes inhibit TGF-ß1 binding. This point is demonstrated in the present investigation by reacting binary α2M-thrombin with trypsin. The inhibition of TGF-ß1 binding caused by proteinases may be steric in nature (the proteinase obstructs the TGF-ß1 binding site). Alternatively, proteinases may cause subtle changes in the structure of the α2M subunits, which decrease TGF-ß1 binding affinity. Future studies should include a complete characterization of the TGF-ß1 binding site in α2M and a determination of the biologic significance of α2M-proteinase-cytokine interactions. Acknowledgment. These studies were supported by the Council for Tobacco Research, (grant no. 2876) and the PEW Scholars Program in the Biomedical Sciences. J. L. is a fellow of the Medical Research Council of Canada.

REFERENCES 1. Fenton JW II: Regulation of thrombin generation and functions. Semin Thromb Hemost 14:234-240, 1988. 2. Shuman MA: Thrombin-cellular interactions. Ann NY Acad Sci 485:228-239, 1986. 3. Fenton JW II: Thrombin bioregulatory functions. Adv Clin Enzymol 6:186-193, 1988. 4. Vu T-KH, DT Hung, VI Wheaton, SR Coughlin: Molecular clon­ ing of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057-1068, 1991.

5. Liu L-W, T-KH Vu, CT Esmon, SR Coughlin: The region of the thrombin receptor resembling hirudin binds to thrombin and alters enzyme specificity. J Biol Chem 266:16977-16980, 1991. 6. Lyons RM, J Keski-Oja, HL Moses: Proteolytic activation of la­ tent transforming growth factor-ß from fibroblast-conditioned medium. J Cell Biol 106:1659-1665, 1988. 7. Massague J: The transforming growth factor-ß family. Annu Rev Cell Biol 6:597-641, 1990. 8. Sottrup-Jensen L: α2-Macroglobulin and related thiol ester plasma proteins. In: Putnam FW: The Plasma Proteins, vol. V. Academic Press, Orlando FL, 1987, pp 191-291. 9. O'Connor-McCourt MD, LM Wakefield: Latent transforming growth factor-ß in serum. A specific complex with α2-macroglobulin. J Biol Chem 262:14090-14099, 1987. 10. Huang SS, P O'Grady, JS Huang: Human transforming growth factor-ß-α2-macroglobulin complex is a latent form of transform­ ing growth factor p. J Biol Chem 263:1535-1541, 1988. 11. LaMarre J, GK Wollenberg, SL Gonias, MA Hayes: Reaction of α2-macroglobulin with plasmin increases binding of transforming growth factors ß1 and ß2. Biochim Biophys Acta 1091:197-204, 1990. 12. Danielpour D, MB Sporn: Differential inhibition of transforming growth factor ß1 and ß2 activity by α2-macroglobulin. J Biol Chem 265:6973-6977, 1990. 13. LaMarre J, MA Hayes, GK Wollenberg, I Hussaini, SW Hall, SL Gonias: An α2-macroglobulin receptor-dependent mechanism for the plasma clearance of transforming growth factor-ß1 in mice. J Clin Invest 87:39-44, 1991. 14. Gonias SL, JA Reynolds, SV Pizzo: Physical properties of human α2-macroglobulin following reaction with methylamine and trypsin. Biochim Biophys Acta 705:306-314, 1982. 15. Barrett AJ, MA Brown, CA Sayers: The electrophoretically "slow" and "fast" forms of human α2-macroglobulin. Biochem J 181:401418, 1979. 16. Pizzo SV, SL Gonias: Receptor mediated protease regulation. In: Conn PM: The Receptors, vol. I. Academic Press, New York, pp 177-221. 17. Moestrup SK, J Gliemann: Purification of the rat hepatic α2-macroglobulin receptor as an approximately 440-kDa single chain pro­ tein. J Biol Chem 264:15574-15577, 1989. 18. Strickland DK, JD Ashcom, S Williams, WH Burgess, M Migliorini, WS Argraves: Sequence identity between the α2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor. J Biol Chem 265:17401-17404, 1990. 19. Hall SW, J LaMarre, LB Marshall, MA Hayes, SL Gonias: Bind­ ing of transforming growth factor-ß1 to α2-macroglobulin reacted

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Radioactivity Recovered

TGF-ß1 -α2M-methylamine

20.

21.

22.

23.

SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 3, 1992 with methylamine and to binary and ternary α2-macroglobulinproteinase complexes. Biochem J in press, 1992. Steiner JP, P Bhattacharya, DK Strickland: Thrombin-induced conformational changes of human α2-macroglobulin: Evidence for two functional domains. Biochemistry 24:2993-3001, 1985. Roche PA, SV Pizzo: Analysis of thiolester bond cleavage-depen­ dent conformational changes in binary α2-microglobulin-proteinase complexes. Arch Biochem Biophys 267:285-293, 1988. Chase T, E Shaw: p-Nitrophenyl-p'-guanidinobenzoate HC1: A new active site titrant for trypsin. Biochem Biophys Res Commun 29:508-514, 1967. Imber MJ, SV Pizzo: Clearance and binding of two electrophoretic

"fast" forms of human α2-macroglobulin. J Biol Chem 256:81348139,1981. 24. Assoian RK: Purification of type-(3 transforming growth factor from human platelets. Methods Enzymol 146:153-163, 1987. 25. Ruff E, A Rizzino: Preparation and binding of radioactively la­ beled porcine transforming growth factor type p. Biochem Bio­ phys Res Commun 138:714-719, 1986. 26. Wakefield LM, TS Winokur, RS Hollands, K Christopherson, AD Levinson, MB Sporn: Recombinant latent transforming growth factor ß1 has a longer plasma half-life in rats than active transforming growth factor (31 and a different tissue distribution. J Clin Invest 86:1976-1984, 1990.

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Effect of human alpha-thrombin on the transforming growth factor-beta 1-binding activity of human alpha 2-macroglobulin.

SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 3, 1992 Effect of Human α-Thrombin on the Transforming Growth Factor-ß1 -Binding Activity of Hum...
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