THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc

Vol. 260. No. 4. Issue of February 25, pp. 2218-2225 1985 Printed in Lj.S.A.

Reactive Site Peptide Structural Similarity between Heparin Cofactor I1 and Antithrombin 111”

(Received for publication, August 22, 1984)

Michael J. Griffith, Claudia M. Noyes, and Frank C. Church From the Departments of Pathology and Medicine, Center for Thrombosis and Hemostasis, The University of North Carolina, Chapel Hill, North Carolina 27514-

Heparin cofactor I1 (Mr = 65,600) was purified 1800-fold from human plasma to further characterize the structural and functional properties of the protein as they compare to antithrombin I11 (Mr = 56,600). Heparin cofactor I1 and antithrombin 111 are function- ally similar in that both proteins have been shown to inhibit thrombin at accelerated rates in the presence of heparin. There was little evidence for structural homology between heparin cofactor I1 and antithrom- bin I11 when high performance liquid chromatography- tryptic peptide maps and NHz-terminal sequences were compared. A partially degraded form of heparin cofac- tor I1 was also obtained in which a significant portion (Mr = 8,000) of the NH2 terminus was missing. The rates of thrombin inhibition (fheparin) by native and partially degraded-heparin cofactor I1 were not signif- icantly different, suggesting that the NHz-terminal re- gion of the protein is not essential either for heparin binding or for thrombin inhibition. A significant de- gree of similarity was found in the COOH-terminal regions of the proteins when the primary structures of the reactive site peptides, i.e. the peptides which are COOH-terminal to the reactive site peptide bonds cleaved by thrombin, were compared. Of the 36 resi- dues identified, 19 residues in the reactive site peptide sequence of heparin cofactor 11 could be aligned with residues in the reactive site peptide from antithrombin 111. While the similarities in primary structure suggest that heparin cofactor I1 may be an additional member of the superfamily of proteins consisting of antithrom- bin 111, al-antitrypsin, al-antichymotrypsin and oval- bumin, the differences in structure could account for differences in protease specificity and reactivity to- ward thrombin. In particular, a disulfide bond which links the COOH-terminal (reactive site) region of an- tithrombin I11 to the remainder of the molecule and is important for the heparin-induced conformational change in the protein and high affinity binding of heparin does not appear to exist in heparin cofactor 11. This observation provides an initial indication that while the reported kinetic mechanisms of action of heparin in accelerating the heparin cofactor II/throm- bin and antithrombin III/thrombin reactions are simi- lar, the mechanisms and effects of heparin binding to the two inhibitors may be different.

Antithrombin 111 is a plasma protease inhibitor which plays

* This work was supported by National Institutes of Health Grants HL-06350, HL-26309, and HL-32656. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

an important role in the regulation of coagulation. Antithrom- bin III/protease reactions result in the formation of stable 1:1 molar complexes in which the active site of the protease is physically blocked by antithrombin 111 (1). Thrombin inhi- bition by antithrombin I11 follows a two-step reaction mech- anism (2) which, at antithrombin I11 concentrations less than 0.1 mM, appears to be a bimolecular process with an apparent second-order rate constant of -5 X lo5 M” min” (2-4). At higher antithrombin 111 concentrations, however, apparent saturation kinetics are observed indicating that formation of an intermediate antithrombin 111-thrombin complex (Kd = 1.4 mM) precedes stable complex formation (2). The chemical nature of the bond which is formed to stabilize the interme- diate complex has not been established; however, it is likely to be an unusually stable tetrahedral adduct (5) or an acyl bond (6-8). Dissociation of various antithrombin 111-protease complexes under alkaline conditions results in cleavage of the reactive site peptide bond (Arg 385-Ser 386) of antithrombin I11 and inactivation of the inhibitor (8, 9). The reactive site peptide, which is COOH-terminal to the bond cleavage site of antithrombin 111, shows significant sequence homology to the reactive site peptide of al-antitrypsin (10, ll), suggesting that the inhibitory mechanisms for both proteins are similar (12).

Many antithrombin III/protease reactions are accelerated by heparin (13), and, until recently, the heparin cofactor activity in plasma, which is required for the anticoagulant effect of heparin (14), appeared to be attributable to anti- thrombin 111 (15). Evidence for a second heparin cofactor activity in plasma (termed heparin cofactor A) was originally reported by Briginshaw and Shanberge (16, 17) and conclu- sively demonstrated more recently in studies reported by Tollefsen and co-workers (18, 19) and others (20-23). Isola- tion of the second heparin cofactor in purified form was first reported by Tollefsen and co-workers and termed heparin cofactor I1 (19) and subsequently reported by Wundenvald and co-workers and termed antithrombin BM (20). As with the antithrombin III/thrombin reaction, the heparin cofactor II/thrombin reaction is accelearated by heparin (17, 19, 20), and kinetic studies have suggested that the general mecha- nism of action of heparin in accelerating both reactions is similar (24). The heparin cofactor II/thrombin reaction also appears to result in the formation of a stable 1:l molar complex (19); however, dissociation of the complex has not been reported. While heparin cofactor I1 is clearly distin- guished from antithrombin 111 in terms of size, immunological reactivity, protease specificity, and apparent affinity for hep- arin (16-24), the general functional similarities between the two heparin cofactors suggest that there might be regions of limited structural homology corresponding to specific func- tional domains.

The present investigation was undertaken to further char-

2218

StructurelFunction of Heparin Cofactor II 2219 TABLE I11

primary structure comparison of heparin cofactor II and antithrombin III

Amino-terminal regions

1 2 3 4 5 6 7 8 9 1 0 11 12 13 14 15 16

H C I I ’ G l y S e r L y s G l y P r o Leu Asp Gln Leu Glu L y s Gly Gly Glu Thr Ala A T I I I b H i s G l y Ser Pro Val Asp I l e C y s Thr Ala L y s P ro Arg Asp I l e P ro

17 18 19 20 21 22 23 24

H C I I Gln S e r Ala Asp P r o Gln Trp Glu . . . ATIII Met Asn P r o Met C y s I l e Tyr Arg . . .

R e a c t i v e s i t e p e p t i d e r e g i o n s

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

HCII‘ Ser Thr Gln Val Arg Phe Thr Val Asp Arg Pro Phe L e u Phe Leu I l e Tyr Glu ATI I Id Ser Leu A s n P r o Arg Val Thr Ala Asn Arg Pro Phe Leu Val Phe I l e Arg Glu

\ / I I Asn Phe-Lys

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

HCI I AT1 I I

H i s Pro Thr Ser Thr Leu Leu Phe Met G l y Arg Val Ala Asn P r o 5 e r Arg Gln Val Pro Leu Asn Thr I l e I l e Phe Met G l y Arg Val Ala Asn P ro Cys Val L y s

‘Data from present study and Witt, et al. (21). Data from Petersen et al. (10). Data from present study. Residue 1 corresponds t.o Ser 386 of native antithrombin 111 (10).

acterize the structural and functional properties of heparin cofactor I1 as they compare to antithrombin 111. An isolation procedure was developed to purify heparin cofactor I1 1800- fold from human plasma with an overall yield approximately s-fold higher than that obtained previously (19). The results we have obtained indicate that there is significant sequence similarity between heparin cofactor I1 and antithrombin 111 in the carboxyl-terminal regions of the proteins where throm- bin interacts. This observation suggests that heparin cofactor I1 is an additional member of the superfamily of proteins which includes ovalbumin, a,-antitrypsin, cul-chymotrypsin, as well as antithrombin 111, which appear to have a common genetic ancestry (11).

EXPERIMENTAL PROCEDURES’

RESULTS

Structural Properties of Heparin Cofactor II-Heparin co- factor I1 and antithrombin I11 did not appear to be structurally similar when amino-terminal amino acid sequences (Table 111) and HPLC2-tryptic peptide maps (Fig. 6) were compared. Only one tryptic peptide from each protein appeared to elute from the HPLC column in the same position (peptide a in Fig. 6). Amino-terminal sequence analysis of these peptides revealed that they were different, however. The peptide ob- tained from the heparin cofactor 11 digest was a tripeptide (Trp-Gly-Lys) while the peptide obtained from the antithrom-

’ Portions of this paper (including “Experimental Procedures,” part of “Results,” part of “Discussion,” Figs. 1-5, and Tables I and 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-2647, cite the authors, and include a check or money order for $8.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

The abbreviations used are: HPLC, high performance liquid chro- matography; SDS, sodium dodecyl sulfate; TosGlyProArgNaN, N“- p-tosyl-glycyl-L-prolyl-L-arginine-p-nitroanilide; PEG, poly(ethy1ene glycol); TEA, triethanolamine.

bin 111 digest was a pentapeptide (Trp-Val-Ser-(X)-Lys) cor- responding to residues 189-193 of the protein (10). The fourth residue of the peptide from antithrombin I11 is glycosylated asparagine (10) which is not extracted from the sequenator cup.

Degraded heparin cofactor I1 was subjected to amino-ter- minal amino acid sequence analysis and appeared to be miss- ing the amino-terminal region of the native protein. While the amino-terminal amino acid could not be unambiguously identified since three residues (Ser, Asn, Arg) were found on cycle 1, subsequent cycles revealed the following (major) residues: Asp (cycle 2), Trp (cycle 3), Ile (cycle 4), and Pro (cycle 5 ) , which distinguished the amino terminus of degraded heparin cofactor I1 from that of antithrombin 111 (His-Gly- Ser-Pro) and from the amino termini of the reactive site peptides of antithrombin 111 and heparin cofactor I1 (see below). While structural similarities between the amino-ter- minal region(s) of degraded heparin cofactor I1 and anti- thrombin Ill might exist, the heterogeneity of the preparat,ion did not permit further comparison.

Evidence for structural similarities between heparin cofac- tor I1 and antithrombin I11 was obtained when the amino acid sequences of the reactive site peptides were compared. Hep- arin cofactor I1 and thrombin formed a stable complex which could be separated from heparin cofactor I1 by size exclusion chromatography as shown in Fig. 7A. The heparin cofactor 11-thrombin complex (Fig. 4, gel I ) was dissociated by incu- bation at pH 10 (Fig. 4, gel J ) , and thrombin-cleaved heparin cofactor I1 was separated from thrombin as shown in Fig. 7B. Amino-terminal sequence analysis of thrombin-cleaved hep- arin cofactor I1 through six cycles revealed the presence of two sequences with the following residues: Gly, Ser (cycle 1); Ser, Thr (cycle 2); Lys, Gln (cycle 3); Gly, Val (cycle 4); Pro, Arg (cycle 5 ) ; Leu, Phe (cycle 6). From these residues the amino-terminal sequences of native heparin cofactor 11 (Gly- Ser-Lys-Gly-Pro-Leu) and the reactive site peptide (Ser-Thr- Gln-Val-Arg-Phe) were derived. Thrombin-cleaved heparin cofactor I1 was subjected to HPLC as described under “Ex- perimental Procedures,” and a single peptide was obtained

2220 Structure/Function of Heparin Cofactor 11

FIG. 6. HPLC tryptic peptide maps of heparin cofactor I1 and antithrombin 111. Heparin cofactor I1 and antithrombin I11 were reduced and carboxymethylated, digested by incubation with trypsin, and samples (1.1 nmol) subjected to reverse-phase HPLC as described under "Experimental Procedures." Peptide elution (right to left in figure) was monitored by absorbance a t 210 nm (0.2 absorbance unit full scale) and 280 nm (0.04 absorbance unit full scale) with the chart speed set a t 5 min/cm (chart divisions are 1 cm). Panel A, heparin cofactor I1 digest; several Am peaks have been labeled as reference points for comparison to the antithrombin I11 digest. Panel B, antithrombin I11 digest. Panel C, combined trypsin digests of heparin cofactor I1 and antithrombin 111.

(not shown). Equivalent amounts of the peptide were obtained when samples were chromatographed before and after reduc- tion with 2-mercaptoethanol. The first 6 residues of the isolated peptide corresponded to those identified above for the reactive site peptide. The complete amino acid sequence of the isolated peptide is shown in Table I11 along with the reactive site peptide sequence reported for antithrombin 111.

Functional Properties of Heparin Cofactor II-In the pres- ence and absence of heparin, the heparin cofactor II/thrombin and antithrombin III/thrombin reactions followed apparent pseudo first-order kinetics. These results are shown in Fig. 8.

A 150 7.3 SO 1 5

1 I I

0.06 - a 0.05 -

0.04 - 4 1

0

0.03 -

0.02 -

0.01 -

a! p 0.020

30 40 50 60 7 0 80

Fraction Numbw

FIG. 7. Isolation of thrombin-cleaved heparin cofactor 11 by Sephacryl 5-300 column chromatography. Panel A, heparin cofactor 11-thrombin complex was prepared and applied to a 2.5 X 90-cm Sephacryl S-300 column as described under "Experimental Procedures." Fractions (5.5 ml) were collected, measured for absor- bance at 280 nm (O), and assayed for heparin cofactor I1 activity (W) and thrombin (A). Approximately 8.0 nmol of active heparin cofactor I1 (7.4 nmol expected) and 0.15 nmol of thrombin (0.13 nmol ex- pected) were recovered. The estimated recovery of heparin cofactor 11-thrombin complex (based on Am values) was 10.5 nmol (15.8 nmol expected). Fractions 51-55 were pooled and a sample subjected to SDS-polyacrylamide gel electrophoresis (see Fig. 4, gel I). Panel E, the heparin cofactor 11-thrombin complex pool obtained above was concentrated and the complex dissociated by incubation at pH 10 (see Fig. 4, gel H) as described under "Experimental Procedures." The solution (containing approximately 8.0 nmol of complex) was then applied to the S-300 column as above. The recoveries of total protein (based on Am values) and of active thrombin corresponded to approximately 50% of the expected amounts. Fractions 53-60, containing thrombin-cleaved heparin cofactor 11, were pooled.

In the absence of heparin, the heparin cofactor II/thrombin reaction rate (L = 0.0071 min") was approximately 20-fold slower than the antithrombin III/thrombin reaction rate ( k o b . = 0.143 min"). Degraded heparin cofactor I1 appeared to react with thrombin at a slightly greater rate than heparin cofactor I1 in the presence and absence of heparin.

DISCUSSION

Heparin cofactor I1 has been shown previously to form a relatively stable 1:l molar complex with thrombin (19). In the present study we have found that the heparin cofactor II- thrombin complex, like the antithrombin 111-thrombin com- plex (8), can be dissociated by incubation at pH 10. Dissocia- tion of the heparin cofactor 11-thrombin complex resulted in cleavage of the heparin cofactor I1 molecule and an apparent reduction in the apparent molecular weight of the protein as

StructurelFunction of Heparin Cofactor II 2221

10 20 30 40 50 60 0.5 1.0 1.5 2.0 2.5 3.0 Reaction Time (min)

FIG. 8. Thrombin inhibition by heparin cofactor 11 and an- tithrombin 111. Thrombin inhibition by heparin cofactor I1 (01, degraded heparin cofactor I1 (m), and antithrombin 111 (A) was measured in the absence, Panel A , and presence, Panel B, of heparin (10 pg/ml). The final inhibitor and thrombin concentrations were 1.7 x 10" and 1.0 x lo-' M, respectively.

determined by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. While dissociation of the antithrom- bin 111-thrombin complex results in cleavage of the antithrom- bin 111 molecule, the apparent molecular weight of the cleaved protein appears lower by SDS-polyacrylamide gel electropho- resis only when reduced by incubation with 2-mercaptoetha- no1 prior to electrophoresis (8). These observations suggested that a disulfide bond similar to the Cys 239-Cys 422 bond which links the reactive site peptide region of antithrombin I11 to the remainder of the molecule (10) does not exist in heparin cofactor 11. Somewhat surprisingly, however, throm- bin-cleaved heparin cofactor I1 eluted from the size exclusion chromatography column in the same volume as did native heparin cofactor I1 (Fig. 7) and was found to retain the reactive site peptide. The isolated thrombin-cleaved heparin cofactor I1 was inactive, as is thrombin-cleaved antithrombin 111, which provided a preliminary indication that the site of cleavage was related to the reactive site peptide bond of the inhibitor. Separation of the reactive site peptide from the remainder of the heparin cofactor I1 molecule was aceom- plished by reverse-phase HPLC, and subsequent amino-ter- minal sequence analysis left little doubt that the peptide was indeed generated by thrombin cleavage at the reactive site peptide bond. Of the 36 residues identified in the reactive site peptide from heparin cofactor I I , l9 residues could be aligned with residues in the reactive site peptide sequence of anti- thrombin 111. While the aligned residues were, for the most part, somewhat removed from the reactive site peptide bond, which cruld explain the differences in protease specificity between the two inhibitors (19, 20), the degree of sequence similarity between the two proteins suggests that they have diverged from a common ancestral gene. Previous studies have suggested that antithrombin I11 belongs to the superfam- ily of proteins consisting of al-antitrypsin, al-antichymotryp- sin, and ovalbumin (10-12). The results obtained in the present study suggest that heparin cofactor I1 may be a fifth member of this superfamily.

There was little evidence for structural homology between heparin cofactor I1 and antithrombin I11 in the amino-termi- nal regions of the proteins, as has been previously noted (21). Interestingly, the amino-terminal region of heparin cofactor I1 does not appear to be essential for heparin cofactor I1

function. Partially degraded heparin cofactor 11, which ap- peared to lack the amino terminus (M? = 8000) of the native protein, inactivated thrombin at essentially the same rates as native heparin cofactor I1 in the presence and absence of heparin. While the amino-terminal region of heparin cofactor I1 may be important for other functional activities of the inhibitor, it seems reasonable to conclude that a significant portion of this region of the protein is not required for either heparin binding or for thrombin inhibition.

Kinetic studies have shown that the heparin-catalyzed hep- arin cofactor II/thrombin and antithrombin III/thrombin re- actions can be described by a rate equation which is function- ally analogous to that describing a random order bireactant enzyme-catalyzed reaction (24). This observation suggests that the interactions of heparin with the two inhibitors may be similar in terms of the catalytic mechanism of action of heparin. The actual mechanism involved in the binding of heparin to heparin cofactor 11, however, may be different from that determined for the binding of heparin to antithrombin I11 which has been shown to be a two-step process (44). The initial rapid equilibrium binding of heparin to antithrombin 111 (Kd = 4.3 X M) induces a conformational change in the antithrombin I11 molecule which increases the overall apparent affinity for heparin by two to three orders of mag- nitude (44). The heparin-induced conformational change in antithrombin 111, detected by an increase in the intrinsic fluorescence (45-481, is abolished by reduction of the Cys 239- Cys 422 disulfide bond, and the high affinity binding of heparin is lost (49, 50). As mentioned earlier (and confirmed by reactive site peptide sequence analysis) a similar disulfide bond does not exist in heparin cofactor 11. Preliminary studies have also shown that the binding of heparin to heparin cofactor I1 does not result in a conformational change in the protein as measured by intrinsic fluorescence (51). These observations suggest that the binding of heparin to heparin cofactor I1 may be a simple one-step process. It can also be noted that the kinetically determined heparin-heparin cofac- tor I1 dissociation constant value is only &fold higher than that determined for heparin-antithrombin I11 (24). This ob- servation may indicate that the functional groups of heparin cofactor I1 required for heparin binding are more accessible in the native conformation of the protein than appears to be the case in antithrombin 111. Further investigation of the structural properties of the heparin binding domains of hep- arin cofactor I1 and antithrombin 111, possibly by comparison of the residues modified in both proteins by pyridoxal 5'- phosphate (52), may provide a better understanding of the interactions of heparin with these two functionally similar proteins and additional insight in the mechanism of action of heparin in catalyzing inhibitor/protease reactions.

Acknowledgments-We would like to thank Dr. Roger L. Lundblad and Lynn Featherstone for their assistance in obtaining the amino acid composition data. We would also like to thank George C. Pan- telakos and Mark O'Neal Speight for their technical assistance.

REFERENCES

1. Harpel, P. C., and Rosenberg, R. D. (1976) Prog. Hemostasis

2. Olson, S. T., and Shore, J. D. (1982) J. Biol. Chern. 257, 14891-

3. Jesty, J. (1979) J. Biol. Chern. 254,10044-10050 4. Griffith, M. J. (1979) J. Biol. Chem. 254, 12044-12049 5. Griffith, M. J., and Lundblad, R. L. (1981) Biochemistry 20,105-

6. Owen, W. G. (1975) Biochim. Biophys. Acta 405,380-387 7. Owen, W. G., Penick, G. D., Yoder, E., and Poole, B. L. (1976)

Thromb. 3 , 145-189

14895

110

Thromb. Haemostasis 35,87-95

2222 StructurelFunction of Heparin Cofactor II 8. Fish, W. W., Orre, K., and Bjork, I. (1979) FEBS Lett. 98, 103- Fenton, J. W., I1 (1976) Thromb. Res. 8,881-886

9. Jornvall, H., Fish, W. W., and Bjork, I. (1979) FEBS Lett. 106, Biochem. 60,149-152 106 29. March, S. C., Parikh, I., and Cuatrecasas, P. (1974) Anal.

30. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412 10. Petersen, T. E., Dudek-Wojciechowska, G., Sottrup-Jensen, L., 31. Griffith, M. J., and Marbet, G. A. (1983) Biochem. Biophys. Res.

and Magnusson, S. (1979) in The Physiological Inhibitors of Commun. 112,663-670 Blood Coagulation and Fibrinolysis (Collen, D., Wiman, B., and 32. Tollefsen, D. M., Pestka, C. A., and Monafo, W. J. (1983) J. BWl. Verstraete, M., e&) pp. 43-54, Elsevier/North-Holland, Am- Chem. 258,6713-6716 sterdam 33. Moore, S. (1963) J. Biol. Chem. 238, 235-237

11. Chandra, T., Stackhouse, R., Kidd, V. J., Robson, K. J. H., and 34. Bredderman, P. J. (1974) Anal. Biochem. 61, 298-301 Woo, S. L. C. (1983) Biochemistry 22, 5055-5061 35. Crestfield, A. M., Stein, W. H., and Moore, S. (1963) J. Biol.

12. Mahoney, W. C., Kurachi, K., and Hermodson, M. A. (1980) Eur. Chem. 238,2413-2419 J. Biochem. 105,545-552 36. Schroeder, W. A., Shelton, J. B., and Shelton, J. R. (1981) in

13. Rosenberg, R. D. (1977) Fed. Proc. 36,lO-18 Advances in Hemoglobin Analysis (Hanash, S. M., and Brewer, 14. Brinkhous, K. M., Smith, H. P., Warner, E. D., and Seegers, W. G. J., eds) pp. 1-22, Alan R. Liss, Inc., New York, NY

15. Abildgaard, U. (1968) Scand. J. Clin. Lab. Invest. 21,89-91 38. Brauer, A. W., Margolies, M. N., and Haber, E. (1975) Biochem- 16. Briginshaw, G. F., and Shanberge, J. N. (1974) Arch. Biochem. istry 14,3029-3035

Biophys. 161, 683-690 39. Tarr, G. E., Beecher, J. F., Bell, M., and McKean, D. J. (1978) 17. Briginshaw, G. F., and Shanberge, J. N. (1974) Thromb. Res. 4, Anal. Biochem. 84, 622-627

463477 40. Klapper, D. G., Wilde, G. E., 111, and Capra, J. D. (1978) A d . 18. Tollefsen, D. M., and Blank, M. K. (1981) J. Clin. Invest. 68, Biochem. 85, 126-131

19. Tollefsen, D. M., Majerus, D. W., and Blank, M. K. (1982) J. 42. Kurachi, K., Schmer, G., Hermodson, M. A., Teller, D. C., and

20. Wunderwald, P., Schrenk, W. J., and Port, H. (1982) Thromb. 43. Nordenman, B., Nystrom, C., and Bjork, I. (1977) J. Biochem.

21. Witt, I., Kaiser, C., Schrenk, W. J., and Wunderwald, P. (1983) 44. Olson, S. T., Srinivasan, K. R., Bjork, I., and Shore, J. D. (1981)

22. Griffith, M. J., Carraway, T., White, G. C., and Dombrose, F. A. 45. Villanueva, G. B., and Danishefsky, I. (1977) Biochem. Biophys.

23. Friberger, D. M., Egberg, N., Holmer, E., Hellgren, M., and 46. Nordenman, B., Danielsson, A., and Bjork, I. (1978) Eur. J. Blomback, M. (1982) Thromb. Res. 25,433-436 Biochem. 90,l-6

24. Griffith, M. J. (1983) P m . Natl. Acad. Sci. U. S. A. 80, 5460- 47. Nordenman, B., and Bjork, I. (1978) Biochemistry 17,3339-3344 5464 48. Villanueva, G., and Danishefsky, I. (1979) Biochemistry 18,810-

25. Franza, B. R., Jr., Aronson, D. L., and Finlayson, J. S. (1975) J. 817 Biol. Chem. 250,7057-7068 49. Longas, M. O., Ferguson, W. S., and Finlay, T. H. (1980) J. Biol.

26. Lundblad, R. L., Kingdon, H. S., and Mann, K. G. (1976) Methods Chem. 255,3436-3441 Enzymol. 45, 156-176 50. Ferguson, W. S., and Finlay, T. H. (1983) Arch. Biochem. BZo-

27. Fenton, J. W., 11, Landis, B. H., Walz, D. A., and Finlayson, J. phys. 221,304-307 S. (1977) in Chemistry and Biology of Thrombin (Lundblad, R. 51. Griffth, M. J., Noyes, C. M., Allen, N., and Villanueva, G. B. L., Fenton, J. W., 11, and Mann, K. G., eds) p. 43, Ann Arbor (1984) Fed. Proc. 43, 1963 Science Publishers, Ann Arbor, MI 52. Pecon, J. M., and Blackburn, M. (1984) J. Biol. Chem. 259,935-

358-362

H. (1939) Am. J. Physiol. 125, 683-687 37. Edman, P., and Begg, G. (1967) Eur. J. Biochem. 1,80-91

589-596 41. Noyes, C. M. (1983) J. Chromutogr. 226,451-460

Biol. Chem. 257,2162-2169 Davie, E. W. (1976) Biochemistry 15,368-373

Res. 25, 177-191 (Tokyo) 78, 195-203

Thromb. Res. 32,513-518 J. Bioi Chem. 256,11073-11079

(1983) Blood 61, 111-118 Res. Commun. 74,803-809

28. Wasiewski, W., Fasco, M. J., Martin, B. M., Detwiler, T. C., and 938

Structure/Function of Heparin Cofactor 11 2223

2224 StructurelFun

I l " " " ' 1

0

Hepa

StructurelFunction of Heparin Cofactor 11 2225 TABLE I1

Beparin Cofactor I1 Amino Acid Composition

IBsInm ll0l X Beeiduesla Mol X Reeidueslb Mol Z R e s i d u d C ndecde llo1ecule nolecule

&partic ac id 12.4 63.2 12.3 62.7 12 .5 63.7 Threortine 6.5 33.1 6.8 34.7 6.4 32.6 serine 6.3 32.1 7.3 37.0 5.8 29.5

Pro l ine 5.1 26.0 3.6 18.3 3.6 18.3 Glycine 6.6 33.6 5.5 27.9 5.3 27.0

Bllf-cy.tloe 1.5 7.6 1.0 5.1 1.0 5.1 va l ine 5.9 30.0 5.7 28.9 6.3 32.1

Glutamic acid 10.8 55.0 11.0 56.2 10.6 54.0

Alanine 5.3 27.0 5.1 25.9 5.0 25.5

Methionine 1.9 9.7 3.0 15.1 2.8 14.3 1.01eucine Leucine

6.2 31.6 5.1 25.9 6.3 32.1 7.8 39.7 10.0 50.9 10.6 54.0

Phenylalanine 5.6 28.4 6.2 31.5 6.1 31.1 L.y.ine Bis t id ine

6.5 33.1 6.4 32.4 6.8 34.6

Arginine 3.0 15.3 2.3 11.5 2.8 14.3 5.1 25.9 4.0 20.3 4.5 22.9 1.3 6.4 2.0 10.0 1.9 9.5

Tyro.ine 2.3 11.7 3.0 15.2 2.5 12.7

Tryptophan

.Data from the present etudy. Besiduedmolecule based on the aseumprion of 65.600 g l m o l heparin Cofactor I1 (19).

f r a Tol lefsen. et a l . (19)

%at. from U i t t , et 11. (21).

D I S a l S S I a

cofac to r 11 from hum- plasma has be0 deecribed. The procedure d i f f e r s from In t h e p r e s e n t r e p o r t a p r o c e d u r e f o r t h e p u r i f i c a t i o n o f hepa r in

t h a t d e s c r i b e d by Tol lefmen and coworkers (19) io t h a t t h e p l a s m a is f r a c t i o n a t e d by b a r i u m c i t r a t e a d s o r p t i o n f o r t h e i s o l a t i o n o f t h e v i tamin K d e p e n d e n t p r o t e i n s prior to initiating s t e p s d e n i g n e d f o r t h e I s o l a t i o n of beparin cofactor 11. Ye have also used PKG to f rac t iona te the p lasma a. oppoeed to the .ulfatcbdaxtrsn hatch .d.orptionlelution step described previoudy (19). w h i l e t h e p r o c e d u r e d e s c r i b e d h e r e i n and t h a t d e e c r i b e d by To l l e f sen and coworkers used similar c o l u m n c h r o m a t o g r a p h y s t e p s t o o b t a i n t h e p u r i f i e d protein, we have found t h a t by revermieg the o rder in which the saiorrexchange andheparin-agarose columns are rum t h e e v e r a l l y i e l d o f h e p a r i n C o f a c t o r I1 (baeed on t h e t o t a l A280 o f p u r i f i e d p r o t e i n o b t a i n e d p e r l i t e r of . tarr ing plasma) is iocreaaed approximately 5-fold.