Proc. Natl. Acad. Sci. USA Vol. 82, pp. 6431-6434, October 1985

Inhibition of chymotrypsin by cofactor II (proteinase inhibitor/al-antichymotrypsin// IMI/glycosaminoglycan) FRANK C. CHURCH, CLAUDIA M. NOYES, AND MICHAEL J. GRIFFITH Division of Hematology, Department of Medicine, Center for and Hemostasis, The University of North Carolina, Chapel Hill, NC 27514 Communicated by Ellis B. Cowling, May 31, 1985

ABSTRACT Human heparin cofactor II is a plasma pro- aminoglycans. We report here that heparin cofactor II in- tein that is known to inhibit thrombin. The rate of thrombin hibits chymotrypsin more rapidly than thrombin, but the inhibition by heparin cofactor II is accelerated (21000-fold) in reaction rate is not affected by glycosaminoglycans. the presence ofthe glycosaminoglycans, heparin and . We have found that chymotrypsin Aa is also inhibited by heparin cofactor II with a second-order rate constant value EXPERIMENTAL PROCEDURES of 1.8 x 106 M'1 min' at pH 8.0 and 250C. However, there was no measurable effect of heparin or dermatan sulfate on the Materials. Human plasma heparin cofactor II was isolated rate of chymotrypsin inhibition. -modified heparin as described (13). Chymotrypsin Aa, heparin (ammonium cofactor II showed a comparable percentage loss of both antichymotrypsin and antithrombin activities. Heparin cofac- salt), dermatan sulfate, and succinyl-Ala-Ala-Pro-Phe-p-ni- tor II and chymotrypsin formed a stable complex with a Mr troanilide were obtained from Sigma. Nutritional Biochemi- value near 90,000 when analyzed by NaDodSO4/polyacryl- cals supplied N-acetyl-a-azaphenylalanine p-nitrophenyl es- amide ; this suggests a 1:1 reaction stoichi- ter; Polybrene and 2,3-butanedione were purchased from ometry. The chymotrypsin cleavage site in heparin cofactor II Aldrich. All buffers used in this study contained 0.1% was the same as that for thrombin, and primary structure (wt/vol) polyethylene glycol (Mr = 8000) to limit analysis of the inhibitor showed a P'l-P'8 sequence of Ser-Thr- adsorption to various surfaces. Gln-Val-Arg-Phe-Thr-Val .... The results indicate that, in Enzyme and Inhibitor Assays. Chymotrypsin activity was contrast to a1-antichymotrypsin, which does not inhibit tryp- measured by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide sin-like enzymes, including thrombin, heparin cofactor II can (14). Heparin cofactor II activity was determined by mea- effectively inhibit both thrombin and chymotrypsin. suring the rate of chymotrypsin inhibition in the absence and presence ofheparin or dermatan sulfate. Stock chymotrypsin There are numerous proteinase inhibitors in normal human solutions (100 AM) were dissolved in 1 mM HCl and kept at plasma that have essential roles during , fibrino- 4°C. Inhibition reactions were started by adding chymotryp- lysis, and inflammation: a1-proteinase inhibitor, antithrom- sin (final concentration, -15-35 nM) to a solution containing bin III, a1-antichymotrypsin, Ci inhibitor (inhibitor of the heparin cofactor II (final concentration, about 0.05-2 ,uM) in activated form of the first component of complement), 50 mM triethanolamine acetate, pH 8.0/100 mM NaCl. a2- inhibitor, and a2- (1). Heparin Portions (0.1 ml) were removed at intervals, added to a cofactor II, described in 1974 by Briginshaw and Shanberge substrate solution (0.8 ml) containing 100 AM succinyl-Ala- (2, 3) and recently purified by Tollefsen et al. (4), is a Ala-Pro-Phe-p-nitroanilide in 50 mM triethanolamine ace- (Mr = 65,600) that functionally resembles anti- tate, pH 8.0/100 mM NaCl, and incubated at ambient thrombin III in the ability to inhibit thrombin at an acceler- temperature. Polybrene (0.5 mg/ml) was included in the ated rate in the presence of heparin. Heparin cofactor II is substrate solution when immunologically distinct from antithrombin III and other inhibition assays were performed in inhibitors (4-7); thus, it can be included with the plasma the presence of heparin or dermatan sulfate. Substrate proteinase inhibitors listed above. Although heparin cofactor hydrolysis was terminated after a suitable time, usually 3-5 II has some structural and functional properties similar to min, by the addition of glacial acetic acid (0.1 ml). p- antithrombin III, the inhibitor exhibits a remarkable speci- Nitroaniline released during the assay was measured at 400 ficity for thrombin (2-7). and other trypsin-like nm and the amount of substrate hydrolyzed was proportional proteinases, including factor Xa, factor IXa, and plasmin, to the chymotrypsin concentration. Antithrombin activity of with a P1 substrate specificity for arginyl or lysyl side chains heparin cofactor II was determined as described (13, 15). [according to the nomenclature of Schechter and Berger (8)], Under the reaction conditions used in which heparin are not inhibited by heparin cofactor II (2-7). cofactor II concentration was in excess of the chymotrypsin We have recently determined that the reactive-site se- concentration, apparent pseudo-first-order kinetics was fol- quence (P1-P'l) in heparin cofactor II is Leu-Ser (9). There- lowed. Under these circumstances, the rate of disappearance fore, the reactive-site sequence in heparin cofactor II resem- of free chymotrypsin (E) may be described by bles that found in a1-antichymotrypsin (10) but not that in antithrombin III, which is Arg-Ser (11). Chymotrypsin Aa has ln(E/Eo) = -k2(IO) t, [11 a substrate specificity primarily directed toward P1 residues that have large or aromatic hydrophobic side where the residual activity (%) is E/Eo at time t, I0is the initial chains (12). The present investigation was undertaken to concentration of heparin cofactor II, and k2 is the second- determine whether or not heparin cofactor II inhibits chy- order rate constant value for reaction of chymotrypsin with motrypsin and if the reaction rate is enhanced by glycos- the inhibitor. Apparent pseudo-first-order rate constant val- ues (kobJ) can be obtained from the slope of ln(E/E0) against The publication costs of this article were defrayed in part by page charge t plots by using various heparin cofactor II concentrations. payment. This article must therefore be hereby marked "advertisement" The kinetic data for chymotrypsin inhibition by heparin in accordance with 18 U.S.C. §1734 solely to indicate this fact. cofactor II were analyzed by the procedure of Kitz and 6431 Downloaded by guest on September 25, 2021 6432 Biochemistry: Church et al. Proc. Natl. Acad. Sci. USA 82 (1985) Wilson (16). The reaction scheme is expressed by

KI k3 IO + E¢i± E-+I-E, [2] 60 where IPE represents a reversible inhibitor-enzyme complex E that is subsequently stabilized, possibly by bond formation, .40- to I-E. When Io>> E, a double reciprocal plot ofkobs values against heparin cofactor II concentration gives Co 1/kobs = (KI/k3)'(1I/o) + l/k3. [3] 620- E The second-order rate constant value for inactivation of chymotrypsin is obtained from the slope and is equivalent to k3/KI, and K1 and k3 values are derived from the x and y 100 30 80 90 120 150 intercepts, respectively (16). Time, sec HPLC and Primary Structure Analysis of Chymotrypsin Hydrolyzed-Heparin Cofactor II. Heparin cofactor II (20 /iM) was incubated with chymotrypsin (8.4 juM) in 50 mM triethanolamine acetate, pH 8.0/100 mM NaCl for 30 min at 250C. The solution was assayed for residual chymotrypsin activity and then was subjected to reverse-phase HPLC on a Vydac C18 TP column (4.6 mm x 25 cm). The column was equilibrated in 0.1% trifluoroacetic acid, and a gradient with 2-propanol to 22% in 57 min and then to 60%o in 34 min was developed at 45°C with a flow rate of 1 ml/min. Heparin cofactor II and chymotrypsin control solutions were also prepared. Amino-terminal sequences were determined on the 0 2 4 6 8 10 eluted . Automated Edman degradation (17) (Beck- 1/Hepann cofactor 11, AM-1 man model 890C protein sequencer) was performed in 0.1 M FIG. 1. Chymotrypsin inhibition by heparin cofactor II. (A) The Quadrol as the coupling buffer with Polybrene as a carrier enzyme (21 nM) was incubated with 0.2 (o), 0.4 (n), and 0.6 (A) ,AM (18). The resulting phenylthiohydantoin amino acids were heparin cofactor II at pH 8.0 and 250C in 50 mM triethanolamine/100 identified by HPLC as detailed earlier (19). mM NaCl buffer. Portions were withdrawn at intervals and assayed Gel Electrophoresis. Complex formation between heparin for remaining activity compared to a control sample (*). (B) Double cofactor II and chymotrypsin was demonstrated by NaDod- reciprocal plot of kob, values against heparin cofactor II concentra- S04/polyacrylamide gel electrophoresis by using 10% tion (obtained at several inhibitor concentrations ranging from 0.1 to acrylamide with the Laemmli buffer system (20). Samples 2.0 AM). were treated at 100°C for 3 min in 1% NaDodSO4. Standards for the calibration of gels were obtained from Bio-Rad (high using the procedure of Levy et al. (24). The slope of a plot of molecular weight protein kit). the logarithm of kobS values against the logarithm of heparin Other Determinations. Protein concentration was deter- cofactor II concentration was 0.9. Thus, :1 mol of heparin mined by using a specific absorption coefficient value of cofactor II inhibited 1 mol of chymotrypsin. 0.915 ml mg-l cm-1 at 280 nm (13) for human heparin In the absence of glycosaminoglycans, heparin cofactor II cofactor II and a Mr = 65,600 (4). Chymotrypsin (Mr = is a more effective inhibitor ofchymotrypsin than ofthrombin 25,000) concentrations were determined at 280 nm with a (Table 1), We tested the ability of heparin and dermatan value of 2.05 mlmg-1cm-1 (12). Active site titration of sulfate to accelerate chymotrypsin inhibition by heparin chymotrypsin was measured with N-acetyl-a-azaphenylala- cofactor II. Under the standard buffer conditions (50 mM nine ester as the substrate the p-nitrophenyl (21) following triethanolamine acetate, pH 8.0/100 mM NaCl), we did not procedure of Gupton et al. (22). Spectrophotometric mea- find an enhanced rate of inhibition in the presence of either surements were performed with a Hewlett Packard 8451A spectrophotometer. Arginine-modified heparin cofactor II heparin or dermatan sulfate with a 2- to 5-fold molar excess of to inhibitor. was prepared by using 10 mM 2,3-butanedione in 50 mM glycosaminoglycan sodium borate (pH 8.0) for 30 min in the dark at 300C (23). We have recently found that modification of arginine residues in heparin cofactor II with 2,3-butanedione results in a loss of antithrombin activity (23). Arginine-modified RESULTS heparin cofactor II lost a proportional percentage of antichy- Properties of Chymotrypsin Inhibition. Incubation of chymotrypsin with heparin cofactor II at pH 8.0 and 250C Table 1. Apparent second-order rate constant values determined resulted in a time-dependent loss of proteinase activity that for proteinase inhibition by heparin cofactor II, followed a first-order process. The semilogarithmic plots of a1-antichymotrypsin, and antithrombin III residual chymotrypsin activity against time at various inhib- k2, itor concentrations were linear to about 10-15% residual Inhibitor Proteinase M-lnmin-1 Ref. activity (Fig. LA). A double reciprocal plot of k*bS values against heparin cofactor II concentration yielded a second- Heparin cofactor II Chymotrypsin 1.8 x 106 order rate constant value of 1.8 x 106 M-1 min-' (Fig. 1B). Thrombin* 5.0 x 104 13 The y-intercept value of the plot approaching zero implied a1-Antichymotrypsin Chymotrypsin 3.6 x 106 25 x 25 that if a reversible intermediate is formed, the interaction is Cathepsin G 3.1 109 III Thrombin* 3.4 x 26 very weak prior to complex stabilization (16). The stoichi- Antithrombin 10W ometry (reaction order) of chymotrypsin inhibition with Trypsin 1.2 x 107 27 respect to heparin cofactor II concentration was estimated by *In the absence of heparin. Downloaded by guest on September 25, 2021 Biochemistry: Church et al. Proc. Natl. Acad. Sci. USA 82 (1985) 6433 by HPLC revealed the elution of one major peak (designated 2) and three minor peaks (designated peptides 1, 3, and 4) (Fig. 2). Peptide 2 was further analyzed by amino- terminal sequencing, and through eight cycles it had the sequence of Ser-Thr-Gln-Val-Arg-Phe-Thr-Val ... . This is the same sequence obtained by thrombin cleavage of heparin cofactor II and corresponds to the carboxyl-terminal peptide (36 residues) of the protein (13). Peptides 3 and 4 were found to correspond to residual heparin cofactor II and undissoci- qlNCM ated complex. Sequence data for peptide 1 were not deter- mined. The extremely small peaks shown in the chromato- gram from approximately 40 to 55 min were attributed to both chymotrypsin and heparin cofactor II (determined from the control solutions). To Mixtures of heparin cofactor II and chymotrypsin were analyzed by NaDodSO4/polyacrylamide gel electrophoresis. l I The formation of a complex with a Mr near 90,000 was 0 40 50 60 70 80 observed, suggesting a 1:1 reaction stoichiometry (data not Elution time, min shown). We were unable to detect total complex formation by gel electrophoresis by using the Laemmli buffer system (20). FIG. 2. HPLC peptide map of the heparin cofactor I-chymo- It has been suggested that this is due to an equilibrium trypsin complex. The inhibitor was allowed to react with chymo- between NaDodSO4-stable and labile proteinase inhibi- trypsin and a portion (2.8 nmol) was analyze(d by reverse-phase tor-enzyme complexes (28). HPLC. Peptide elution was monitored at 210 nm (0.5 absorbance unit full scale) and 280 nm (0.05 absorbance unit full scale). DISCUSSION motrypsin activity relative to antithrombin activity. The kobs In the present study we have shown that chymotrypsin Aa is values of modified heparin cofactor II for chymotrypsin rapidly inhibited by heparin cofactor II and that the reaction (inhibitor:proteinase was 0.4 liM:21 nM) and thrombin (in- follows apparent second-order kinetics. Apparent second- hibitor:proteinase was 0.3 ,tM:4 nM) inhibition were 30% and order rate constant values for inhibition of chymotrypsin and 25%, respectively, of the control sample not treated with other proteinases by heparin cofactor II, a1-antichymotryp- 2,3-butanedione (kobs values for antichymotrypsin and anti- sin, and antithrombin III are compared in Table 1. Thrombin thrombin activities were 0.68 min- and 0.016 min', respec- and chymotrypsin react with the same site on heparin tively). The results suggest that there is an arginine residue(s) cofactor II and both appear to form 1:1 molar complexes with in heparin cofactor II that is essential for both antichymo- the inhibitor. Therefore, heparin cofactor II is a "single- trypsin and antithrombin activities. headed" proteinase inhibitor (at least with respect to throm- Interaction of Heparin Cofactor II with Chymotrypsin. bin and chymotrypsin) as opposed to "multi-headed" as has Evidence for a similar cleavage site (reactive-site bond) in been described for other proteinase inhibitors (29). heparin cofactor II between chymotrypsin and thrombin was Hunt and Dayhoff (30) proposed a new examined. We previously determined the primary sequence consisting of (31), antithrombin III (32-34), and of the reactive-site peptide, beginning with the P'1 residue, a1-proteinase inhibitor (35, 36) (Fig. 3). a1-Antichymotrypsin released from the inhibitor-thrombin complex and purified (10, 37), angiotensinogen (38), and heparin cofactor II (13) by reverse-phase HPLC (13). have recently been suggested to belong in this family. Analysis ofthe heparin cofactor 1I-chymotrypsin complex Heparin cofactor II and a1-antichymotrypsin have some

Pi-P 1 P 5 P 10 P 15 Heparin Cofactor II Leu Ser Thr Gln Val Arg Phe Thr Val Asp Arg Pro Phe Leu Phe Leu Ile Ovalbumin Ala Ser Val Ser Glu Glu Phe Arg Ala Asp His Pro Phe Leu Phe Cys Ile Antithrombin III Arg Ser Leu Asn Pro Asn Arg Val Thr Phe Lys Ala Asn Arg Pro Phe Leu Val Phe Ile al-Proteinase Inhibitor Met Se Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met t 1-Antichymotrypsin Leu Ser Ala Leu Val Glu Arg Thr Ile Val Arg Phe Asn Arg Pro Phe Leu Met lie Ile Thr

P 20 P 25 p 30 P 35 Heparin Cofactor II Tyr Glu His Pro Thr Ser Thr Ile Ile Phe Net Gly Arg Val Ala Asn Pro Ser Arg Gln Ovalbumin Lys His Ile Ala Thr Asn Ala Val Leu Phe Phe Gly Arg Cys Val Ser Pro Antithrombin III Arg Glu Val Pro Leu Asn Thr Ile Ile Phe Met Gly Arg Val Ala Asn Pro Cys Val Lys al-Proteinase Inhibitor Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr Gln Lys aXi-Antichymotrypsin Val Pro Thr Asp Thr Gln PheI Phe Met Ser Lys Val Thr Asn Pro Ser Lys Pro Asn-IleI FIG. 3. Reactive-site peptide sequence of heparin cofactor II aligned with that of ovalbumin, antithrombin III, a1-proteinase inhibitor, and a1-antichymotrypsin. The reactive-site bonds in all of the inhibitors are located in the carboxyl-terminal ends of the . The P1 residue for each protein corresponds to the following amino acid residue found in the total protein sequence (given in parentheses): ovalbumin (Ala-353) (31), antithrombin III (Arg-393) (32-34), a1-proteinase inhibitor (Met-358) (35, 36), a1-antichymotrypsin (Leu-358) (10, 37), and heparin cofactor II (full sequence not known) (present study; refs. 9, 13). The reactive-site residues (Pl-P'l) are enclosed in the box. Downloaded by guest on September 25, 2021 .:"34,C A Biochemistry: Church et al. Proc. Natl. Acad. Sci. USA 82 (1985) sequence in the reactive-site peptide region (Fig. M. C. (1979) Anal. Biochem. 99, 316-320. 3). However, unlike heparin cofactor II, a1-antichymotryp- 15. Church, F. C. & Griffith, M. J. (1984) Biochem. Biophys. Res. sin, which inhibits chymotrypsin-like proteinases, is not Commun. 124, 745-751. reactive toward any trypsin-like enzymes (1). Although the 16. Kitz, R. & Wilson, I. B. (1962) J. Biol. Chem. 237, 3245-3249. 17. Edman, P. & Begg, G. (1967) Eur. J. Biochem. 1, 80-91. three-dimensional structure is not known, the results of the 18. Tarr, G. E., Beecher, J. F., Bell, M. & McKean, D. J. (1978) present investigation imply that the reactive-center region in Anal. Biochem. 84, 622-627. heparin cofactor II is able to recognize both thrombin and 19. Noyes, C. M. (1983) J. Chromatogr. 226, 451-460. chymotrypsin. 20. Laemmli, U. K. (1970) Nature (London) 227, 680-685. Thrombin inhibition by heparin cofactor II is very rapid in 21. Elmore, D. T. & Smyth, J. J. (1968) Biochem. J. 107, 103-107. the presence of heparin or dermatan sulfate with second- 22. Gupton, B. F., Caroll, D. L., Tuhy, P. M., Kam, C.-M. & order rate constant values approaching ca. 108 M-1 min'- (4, Powers, J. C. (1984) J. Biol. Chem. 259, 4279-4287. 6, 13, 39, 40). Interestingly, there is no apparent enhancement 23. Church, F. C., Noyes, C. M., Tyndall, J. A. & Griffith, M. J. of the rate of chymotrypsin inhibition by heparin cofactor II (1985) Thromb. Haemostasis Gen. Inf. 54, 203. 24. Levy, H. M., Leber, P. D. & Ryan, E. M. (1963) J. Biol. including these glycosaminoglycans. This may be due to the Chem. 238, 3654-3659. inability of chymotrypsin to bind heparin, as does thrombin, 25. Beatty, K., Bieth, J. & Travis, J. (1980) J. Biol. Chem. 255, to form a ternary proteinase-heparin-inhibitor complex (40). 3931-3934. Although it appears that an in vivo role can be proposed for 26. Downing, M. R., Bloom, J. W. & Mann, K. G. (1978) Bio- thrombin inhibition by heparin cofactor 11 (4, 6, 13, 39, 40), chemistry 17, 2649-2653. an additional role related to chymotrypsin-like proteinase 27. Bjork, I. & Lindahl, U. (1982) Mol. Cell. Biochem. 48, inhibition might also be possible. There are numerous 161-182. chymotrypsin-like proteinases suggested to have a physio- 28. Travis, J., Bowen, J. & Baugh, R. (1978) Biochemistry 17, logical role in protein turnover and in tissue-localized 5651-5656. angio- 29. Laskowski, M., Jr., & Kato, I. (1980) Annu. Rev. Biochem. 49, tensin II formation: peritoneal mast cell proteinase I (41), 593-626. skeletal muscle mast cell proteinase 11(42), cytoplasmic mast 30. Hunt, L. T. & Dayhoff, M. 0. (1980) Biochem. Biophys. Res. cell chymase (43), salivary gland tonin (44, 45), skin Commun. 95, 864-871. chymotrypsin-like proteinase (45, 46), and 31. McReynolds, L., O'Malley, B. W., Nisbet, A. D., Fothergill, cathepsin G (28, 45-48).* Although there are differences in J. E., Givol, D., Field, S., Robertson, M. & Brownlee, G. G. the structural, functional, and catalytic activities of these (1978) Nature (London) 273, 723-728. chymotrypsin-like enzymes compared to chymotrypsin 32. Peterson, T. E., Dudek-Wojecieckowska, G., Sottrup-Jensen, (41-48), one ofthese proteinases may be inhibited by heparin L. & Magnusson, S. (1979) in The Physiological Inhibitors of cofactor II or without at a Blood Coagulation and , eds. Collen, D., Wiman, (with glycosaminoglycan) physi- B. & Verstraete, M. (Elsevier/North-Holland, Amsterdam), ologically important rate. pp. 43-54. 33. Prochownik, E. V., Markham, A. F. & Orkin, S. H. (1983) J. *Parker and Tollefsen (49) recently examined the proteinase speci- Biol. Chem. 258, 8389-8394. ficity ofheparin cofactor II. Aside from thrombin, no other enzymes 34. Chandra, T., Stackhouse, R., Kidd, V. J. & Woo, S. L. C. associated with coagulation or fibrinolysis are inhibited. However, (1983) Proc. Natl. Acad. Sci. USA 80, 1845-1848. cathepsin G is inhibited by heparin cofactor II with a k2 value of 8.4 35. Kurachi, K., Chandra, T., Friezner Degen, S. J., White, T. T., x 104 M -min1 in the presence of dermatan sulfate. Marchioro, T. L., Woo, S. L. C. & Davie, E. W. (1981) Proc. Natl. Acad. Sci. USA 78, 6826-6830. 36. Carrell, R. W., Jeppson, J.-O., Laurell, C.-B., Brennan, S. O., This work was supported in part by Research Grants HL-07255, Owen, M. C., Vaughan, L. & Boswell, D. R. (1982) Nature HL-32656, and HL-06350 from the National Institutes of Health. (London) 298, 329-334. 37. Chandra, T., Stackhouse, R., Kidd, V. J., Robson, K. J. H. & 1. Travis, J. & Salvesen, G. S. (1983) Annu. Rev. Biochem. 52, Woo, S. L. C. (1983) Biochemistry 22, 5055-5061. 655-709. 38. Doolittle, R. F. (1983) Science 222, 417-419. 2. Briginshaw, G. F. & Shanberge, J. N. (1974) Arch. Biochem. 39. Tollefsen, D. M., Pestka, C. A. & Monafo, W. J. (1983) J. Biophys. 161, 683-690. Biol. Chem. 258, 6713-6716. 3. Briginshaw, G. F. & Shanberge, J. N. (1974) Thromb. Res. 4, 40. Griffith, M. J. (1983) Proc. Natl. Acad. Sci. USA 80, 463-477. 5460-5464. 4. Tollefsen, D. M., Majerus, D. W. & Blank, M. K. (1982) J. 41. Everitt, M. T. & Neurath, H. (1979) Biochimie 61, 653-662. Biol. Chem. 257, 2162-2169. 42. Woodbury, R. G. & Neurath, H. (1978) Biochemistry 17, 5. Tollefsen, D. M. & Blank, M. K. (1981) J. Clin. Invest. 68, 4298-4304. 589-596. 43. Schwartz, L. B., Riedel, C., Canfield, J. P., Wasserman, S. I. 6. Griffith, M. J., Carraway, T., White, G. C. & Dombrose, & Austen, K. F. (1981) J. Immunol. 126, 2071-2078. F. A. (1983) Blood 61, 111-118. 44. Boucher, R., Asseleir, J. & Genest, T. (1974) Circ. Res. 34/35, 7. Wunderwald, P., Schrenk, W. J. & Port, H. (1982) Thromb. Suppl. 1, 1203-1209. Res. 25, 177-191. 45. Wintroub, B. U., Schechter, N. M., Lazarus, G. S., 8. Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Kaempfer, C. E. & Schwartz, L. B. (1984) J. Invest. Derma- Commun. 27, 157-162. tol. 83, 336-339. 9. Griffith, M. J., Noyes, C. M., Tyndall, J. A. & Church, F. C., 46. N. J. J. C. & (1985) Biochemistry, in press. Schechter, M., Fraki, E., Geesin, Larazus, G. S. 10. M. & Travis, J. (1983) J. Biol. Chem. 258, 12749-12752. (1983) J. Biol. Chem. 258, 2973-2978. Morii, 47. M. M. K. F. & 11. Jornvall, H., Fish, W. W. & Bjork, I. (1979) FEBS Lett. 106, Tonnesen, G., Klempner, S., Austen, 358-362. Wintroub, B. U. (1982) J. Clin. Invest. 69, 25-30. 12. Wilcox, P. E. (1970) Methods Enzymol. 19, 64-108. 48. Yoshida, N., Everitt, M. T., Neurath, H., Woodbury, R. G. & 13. Griffith, M. J., Noyes, C. M. & Church, F. C. (1985) J. Biol. Powers, J. C. (1980) Biochemistry 19, 5799-5804. Chem. 260, 2218-2225. 49. Parker, K. A. & Tollefsen, D. M. (1985) J. Biol. Chem. 260, 14. DelMar, E. G., Largman, C., Brodrick, J. W. & Geokas, 3501-3505. Downloaded by guest on September 25, 2021