Primary structure of two-chain botrocetin, a von Willebrand factor ...

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FGSN C Y R F F A V S L T W A EEQFCQ S F S V P S R|G|D I D S I GH L V SI H. 47 T CLY. -G WHKF Q Q .... Thrombosis, eds. Bloom, A. L. & Thomas, D. P. (Churchill ... Smith, T. F., Waterman, M. S. (1981) Adv. Appl. Math. 2,. 482-489. 25. Giga ...
Proc. Nati. Acad. Sci. USA Vol. 90, pp. 928-932, February 1993 Biochemistry

Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca (platelet glycoprotein Ib)

YOSHIKO USAMI*t, YOSHIHIRo FUJIMURAt, MASAMI SUZUKI*, YASUHIRO OZEKI*, KENJI NISHIOt, HIROMU FUKUIt, AND KoITI TITANI*§ *Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, School of Medicine, Fujita Health University, Toyoake, Aichi 470-11, Japan; and tDepartment of Blood Transfusion, Nara Medical College, Kashihara, Nara 634, Japan

Communicated by Kenneth M. Brinkhous, October 19, 1992

before and a 15,000/14,500 doublet after reduction (15). On a weight basis, two-chain botrocetin was -30 times more active than the one-chain species in promoting vWF binding to GPIb. N-terminal amino acid sequence analysis of twochain botrocetin showed it to be a heterodimer composed of an a subunit with Mr 15,000 and a ,3 subunit with Mr 14,500. The N-terminal sequence of one-chain botrocetin showed no similarity to that of the two-chain species, suggesting that they are different molecules (15). We now report the complete amino acid sequences of the a and ,B subunits of two-chain botrocetin (which are composed of 133 and 125 amino acid residues, respectively), as well as the location of intra- and interchain disulfide bonds within the molecule.

ABSTRACT The complete amino acid sequence and location of the disulfide bonds of two-chain botrocetin, which promotes platelet agglutination in the presence of von Willebrand factor, from venom of the snake Bothrops jararaca are presented. Sequences of the a and .8 subunits were determined by analysis of peptides generated by digestion of the S-pyridylethylated protein with Achromobacter protease I or a-chymotrypsin and by chemical cleavage with cyanogen bromide or 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Twochain botrocetin is a heterodimer composed of the a subunit (consisting of 133 amino acid residues) and the ,B subunit (consisting of 125 amino acid residues) held together by a disulfide bond. Seven disulfide bonds link half-cystine residues 2 to 13, 30 to 128, and 103 to 120 of the a subunit; 2 to 13, 30 to 121, and 98 to 113 of the ,B subunit; and 80 of the a subunit to 75 of the /3 subunit. In terms of amino acid sequence and disuffilde bond location, two-chain botrocetin is homologous to echinoidin (a sea urchin lectin) and other C-type (Ca2+dependent) lectins.

MATERIALS AND METHODS Purification of Two-Chain Botrocetin. Two-chain botrocetin was purified from crude venom of B. jararaca (Sigma) as recently described (15). Reduction and S-Pyridylethylation of Two-Chain Botrocetin. One milligram of two-chain botrocetin was reduced with 20 ,ul (81 mM) of tri-n-butylphosphine (16) (Wako Pure Chemical, Osaka) and then S-pyridylethylated with 10 I1(94 mM) of 4-vinylpyridine (17) (Tokyo Kasei Kogyo, Japan) in 1 ml of 0.3 M Tris buffer (pH 8.3) containing 6 M guanidine hydrochloride at room temperature for 3 h. The S-pyridylethylated a (PE-a) and P (PE-,B) subunits were separated by reversed-phase HPLC (RP-HPLC) on a SynChropak RP-8 column (4.1 x 250 mm; SynChrome, Lafayette, IN) as shown in Fig. 1. Enzymatic and Chemical Cleavage of PE-a and -(3 Subunits. Each S-pyridylethylated subunit was digested with Achromobacter protease I (API; a generous gift from T. Masaki, Ibaraki University, Japan), which specifically cleaves lysyl bonds (18), at 37°C in 50 mM Tris HCl (pH 9.0) in the presence of 2 M urea, at an enzyme-to-substrate ratio of 1:150 by weight. The PE-a subunit was also digested with a-chymotrypsin (Worthington) in 0.1 M NH4HCO3 at an enzyme-to-substrate ratio of 1:100. Cleavage with cyanogen bromide (Wako Pure Chemical) was performed in 70% formic acid at room temperature as described by Gross (19). Each S-pyridylethylated subunit was cleaved with 2-(2'-nitrophenylsulfenyl)-3-methyl3-bromoindolenine (BNPS-skatole) (Pierce) as described by Omenn et al. (20). Separation of Peptides. Peptides were separated by RPHPLC on a SynChropak RP-8 or Cosmosil 5C8-300 column

von Willebrand factor (vWF) is a multimeric glycoprotein that circulates in blood forming a noncovalent complex with coagulation factor VIII (1, 2). vWF plays a critical role in the platelet adhesion process at high shear stress by binding to the subendothelial matrix at sites of vascular injury (3). To reproduce this event in vitro, the binding of vWF to platelet glycoprotein lb (GPIb) is an essential step. Since the circulating native form of vWF does not bind to platelet GPIb, experimental models of this interaction have been determined in the presence of exogenous modulators, such as the antibiotic ristocetin or after removal of terminal sialic acid residues from carbohydrate side chains of vWF (2-5). In 1978, Read et al. (6) partially purified a vWF-dependent platelet coaggulutinin named botrocetin from the snake venom of Bothrops jararaca. Thereafter, Read et al. (6, 7) and Brinkhous et al. (8-10) have extensively characterized functional aspects of botrocetin. Like ristocetin, botrocetin does induce vWF binding to platelet GPIb (9-12). However, unlike ristocetin, botrocetin reacts with a broad spectrum of multimeric forms of vWF and induces vWF-dependent platelet aggulutination in various animal species (6-10). Recent studies showed that purified botrocetin forms an activated complex with vWF and then binds to GPIb (9-15). Andrews et al. (14) suggested that purified botrocetin is a heterodimer composed of subunits with apparent Mr values of 14,000 and 14,500. Subsequently, we purified two distinct forms of botrocetin: a one-chain species with apparent Mr values of 28,000 before and 32,000 after reduction of disulfide bonds and a two-chain species with apparent Mr values of 27,000

Abbreviations: API, Achromobacter protease I; BNPS-skatole, 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine; GPIb, glycoprotein Ib; PE-a (or -(3) subunit, S-pyridylethylated a (or (8) subunit; RP-HPLC, reversed-phase HPLC; vWF, von Willebrand factor. tPresent address: Department of Pharamceutics, Gifu Pharmaceutical University, Gifu 502, Japan. §To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 928

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(4.6 x 100 mm; Nacalai Tesque, Kyoto) with gradients of acetonitrile in dilute aqueous trifluoroacetic acid (21). Amino Acid Analysis and Sequence Determination. Each intact PE subunit was hydrolyzed in 6 M HCl containing 1% phenol at 1100C for 24 h by the vapor-phase method and analyzed on a Hitachi model L8500 amino acid analyzer. Smaller peptides obtained by enzymatic or chemical cleavage were analyzed by the 4-(dimethylamino)azobenzenesulfonyl chloride method (22) after a 24-h acid hydrolysis. Sequence determination was carried out with an Applied Biosystems model 470A protein sequencer connected to a model 120A phenylthiohydantoin analyzer. Assignment of Disulfide Bonds. Intact two-chain botrocetin was cleaved with cyanogen bromide in 70% (vol/vol) formic acid and lyophilized. The lyophilized sample was further digested with API in 50 mM Tris HCl (pH 9.0) in the presence of 2 M urea as described above. After separation of the digest by RP-HPLC on a SynChrome RP-8 column as described above, cystine-containing peptides were identified by amino acid analysis and subjected to sequence analysis. Sequence Homology Search. Sequence homology was searched in the protein sequence data base of the National Biomedical Research Foundation (March 1991) on a VAX 3600 computer using the WORDSEARCH program (23) (version 6.0, April 1989). The alignment procedure used the SEGMENT program (24).

RESULTS Separation of a and (a Subunits of Two-Chain Botrocetin. Reduced and S-pyridylethylated two-chain botrocetin was separated into four major peaks by RP-HPLC on a SynChropak RP-8 column as shown in Fig. 1. On the basis of N-terminal sequence analysis, peak 2 was identified as the PE-a subunit, and peaks 3 and 4 were identified as the PE-f3 subunit. Peak 1, which yielded no amino acid by amino acid analysis, apparently represented a reagent impurity with UV absorbance at 206 nm. Amino Acid Sequence of the a Subunit. Sequence analysis of the intact PE-a subunit (-250 pmol) yielded an unambiguous N-terminal sequence of 45 residues.

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An API digest of the PE-a subunit (-2 nmol) was separated into nine major fractions by RP-HPLC as shown in Fig. 2, and the peptides were designated as K1-K15. Ten major peptides (not including the small hydrophilic peptides K5, K6, K9, K11, and K15, which appeared to be in the breakthrough peak) were isolated and subjected to amino acid analysis (data not shown) and sequence determination (Fig. 3). Peptides K4 and K14 were isolated and analyzed as a mixture. Their sequences were later identified within fragments M2 (or W3) and W6, respectively. Upon analysis of two cyanogen bromide fragments of the PE-a subunit (=2 nmol) separated in a similar manner by RP-HPLC (data not shown), M2 was found to extend the N-terminal sequence obtained with intact PE-a subunit to Phe-51 and also to provide overlaps of K3-K7. To obtain the remaining overlaps of API peptides, the PE-a subunit (-5 nmol) was cleaved at tryptophanyl bonds with BNPS-skatole. After extraction of excess reagents with 1-chlorobutane, the mixture was separated in a similar manner by RP-HPLC (data not shown). Sequence analysis of peptides W3-W6 thus isolated provided overlaps of API peptides K3-K8, K8-K1O, K10-K13, and K13-K15 (hypothetical C-terminal K peptide), respectively (Fig. 3). Peptide W6 was presumed to be derived from the C terminus of the PE-a subunit, but no sequence was obtained beyond Pro-131. To clarify the C-terminal sequence of the entire subunit, the PE-a subunit was digested with a-chymotrypsin and separated by RP-HPLC. The C-terminal peptide C1 was searched for by amino acid analysis based on high proline content and analyzed to the C-terminal end (Fig. 3). Amino Acid Sequence of the P Subunit. Sequence analysis of the intact PE-f3 subunit (-250 pmol) yielded an N-terminal sequence of 27 residues, with a tentative identification of tryptophan at residue 20 and an ambiguous identification at residue 23. An API digest of the PE-,8 subunit (":2.5 nmol) was separated by RP-HPLC in a manner similar to that of the PE-a subunit (data not shown). Seven major peptides, designated as K1-K8 (K5 and K6 were isolated as an overlapping peptide K5/6), were subjected to amino acid analysis and sequence determination (Fig. 3). Peptide K2 was analyzed to extend the N-terminal sequence obtained with intact PE-,B subunit to Lys45. Upon analysis offour cyanogen bromide fragments of .--T

K3 KJO

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FIG. 1. Separation of a and (8 subunits of two-chain botrocetin. After reduction and S-pyridylethylation, the botrocetin was subjected to RP-HPLC on a SynChropak RP-8 column using a trifluoroacetic acid/acetonitrile system at a flow rate of 2.0 ml/min. Peptides were monitored at 206 nm.

0

10

20

30

40

RETENTION TIME (min) FIG. 2. Primary separation of peptides generated by API digestion of the PE-a subunit. The digest was separated by RP-HPLC as described for Fig. 1. Purified peptides are identified by a K prefix as described in Fig. 3.

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a

20

30

40

50

70

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DCPSGNSSYKGNCYKFFQQUUWADAZERFCSEQAKGGELVSIKIYSKKDFVGDLVTKINQSSDLYAWIG intact El DCPSGWSSYEGNCYKFFQQKMNWADAERFCSEQAKGGHLVSIKIY---

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FIG. 3. Sequence of the a and 8 subunits of two-chain botrocetin. The proven sequences of specific peptides (underlined) are given in one-letter code below the summary sequence. Prefixes K, M, W, and C denote peptides generated by cleavage of the PE-a or PE-N subunit at lysyl, methionyl, and tryptophanyl bonds and by digestion with a-chymotrypsin, respectively. Products of lysyl, methionyl, and tryptophanyl cleavage are numbered from the N terminus toward the C terminus of each subunit. Sequences written in uppercase letters were proven by Edman degradation; those in lowercase letters indicate tentative identifications. Unidentified residues are shown by dashes.

PE-,3 subunit (-2 nmol), M2, M3, and M4 were found to provide overlaps of API peptides K2 and K3; K3, K4, and K5/6; and K5/6 and K7, respectively. Subdigestion of fragment M4 with BNPS-skatole yielded a subpeptide M4-W3, which was found to provide overlap of K7 and K8. Peptide K8 was assigned to the C terminus, because it lacked lysine. Location of Intra- and Interchain Disulfide Bonds of TwoChain Botrocetin. Six cystine-containing peptide fractions were isolated by RP-HPLC following cleavage with cyanogen bromide and API digestion of the intact two-chain botrocetin in a manner similar to that described for PE-a subunit digestion (Fig. 2). One fraction contained two cystinecontaining peptides and was further separated by RP-HPLC on a uBondasphere 5-pum C18 300-A column (3.9 x 150 mm; Nihon Waters, Tokyo). All seven cystine-containing peptides yielded two phenylthiohydantoin derivatives of amino acids in almost equal amounts at each cycle of Edman degradation, indicating that each consisted in turn of two disulfide-linked peptides. Sequence analysis of the seven peptides indicated that disulfide bonds link six half-cystine residues (2 to 13, 30 to 128, and 103 to 120) in the a subunit and six half-cystine residues (2 to 13, 30 to 121, and 98 to 113) in the f3 subunit. One interchain disulfide bond was identified between Cys-80 of the a subunit and Cys-75 of the 1 subunit, indicating that two-chain botrocetin is a heterodimer (Fig. 4). Location of the intrachain disulfide bonds is similar in two-chain botrocetin and echinoidin (a lectin from the sea urchin Anthocidaris crassispina) (25), but the location of the interchain disulfide bond is quite different in the two proteins (Fig. 4). Contrarily, a similarity in the location of the interchain disulfide bonds was found between two-chain botrocetin and the recently described rattlesnake lectin (26) (Fig. 4).

Sequence Homology Search. The amino acid sequence of two-chain botrocetin was compared with known sequences in the protein data base. The sequences of both the a and a =

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FIG. 4. Location of intrachain and interchain disulfide bonds in two-chain botrocetin, echinoidin, and rattlesnake lectin. Numbers indicate positions of cysteine residues in each subunit.

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Proc. Natl. Acad. Sci. USA 90 (1993)

subunits are highly homologous to those of echinoidin (25), human proteoglycan core protein (27), and other C-type (Ca2+-dependent) lectins (26, 28, 29) (Fig. 5).

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values of the a and p subunits (15,191 and 15,014, respectively) are in good agreement with their apparent Mr values of 15,000 and 14,500 as estimated by SDS/PAGE (15). This structural analysis also revealed that two-chain botrocetin is a member of the Ca2+-dependent (C-type) lectin superfamily. Recently, Drickamer (35) reported that various C-type lectins contain 18 invariant amino acid residues within the molecules as examined by sequence homology search. These residues usually generate a C-terminal carbohydraterecognition domain (CRD) capable of binding selectively to carbohydrate affinity columns. The carbohydrate-recognition domain of the a subunit of botrocetin corresponds to the region that contains seven completely conserved residues: Gly-il, Cys-30, Gly-70, Cys-103, Trp-105, Cys-120, and Cys-128. Similarly, conserved residues in the p subunit are as follows: Gly-il, Cys-30, Gly-67, Asp-70, Cys-98, Trp-109, Cys-113, Cys-121, and Glu-122. However, the activity of botrocetin in promoting vWF binding to platelets is not inhibited by EDTA (9, 10) or lactose and other sugars (Y.U., Y.O., and K.T., unpublished data). Therefore, it is unlikely that botrocetin binds to the carbohydrate moiety of vWF via the substrate-binding site of the lectin. At present, it is unclear whether the vWF-binding site of botrocetin resides on the a or 1 subunit, or both, although complete reduction of the disulfide bonds results in a total loss of platelet agglutinating activity (15). Sequence homology search also revealed that the a and ,B subunits of botrocetin have -32.8% and 30.3% sequence identity with sea urchin echinoidin, respectively, but these values are lower than the ==44.8% identity between the subunits themselves. There is no significant similarity between the one-chain and two-chain species, in agreement

DISCUSSION Two-chain botrocetin forms an "activated" complex with vWF, and the complex then binds to platelet GPIb, resulting in platelet agglutination (10). We have demonstrated that an anti-vWF monoclonal antibody, NMC4, and an anti-GPIb monoclonal antibody, AP-1, inhibit both botrocetin- and ristocetin-induced vWF binding to GPIb (30, 31). Our recent results (32) indicate that NMC-4 blocks formation of an activated complex between vWF and two-chain botrocetin, supporting the previous observation that botrocetin binds solely to vWF and not to GPIb (13, 14). Furthermore, neither of two synthetic peptides corresponding to two noncontiguous sequences in the constituent subunits of vWF, Cys-474 to Pro-488 and Leu-694 to Pro-708, which were identified as the ristocetin-dependent GPIb binding domain (33), blocked botrocetin-induced binding. Sugimoto et al. (34) have recently shown more direct evidence that three discontinuous synthetic peptides of vWF subunit, Asp-539 to Val-553, Lys-569 to Gln-583, and/or Arg-629 to Lys-643, block the formation of the vWF-botrocetin complex. These results indicate that ristocetin or botrocetin independently modulates vWF and that their respective GPIb binding domains are not precisely congruent. Since it is well established that intact vWF does not bind spontaneously to GPIb, it seems most likely that binding of botrocetin to vWF initiates a conformational change by which the cryptic GPIb-binding domain of intact vWF is exposed on the surface. Obviously, a better understanding of the structure-function relationships of botrocetin needs to be obtained. Indeed, there are two distinct forms of botrocetin, of which two-chain botrocetin is 30 times more active in promoting vWF binding to GPIb than the one-chain species (15). In the present study, we have elucidated the complete amino acid sequence of two-chain botrocetin and the location of intra- and interchain disulfide bonds within the molecule. Two-chain botrocetin is a heterodimer composed of an a subunit of 133 residues and a, subunit of 125 residues held together by one interchain disulfide bond. Calculated Mr

with our previous report (15). Two-chain botrocetin and echinoidin are very similar in the location of three intrachain disulfide bonds in each subunit, but very different in the location of one interchain disulfide bond. Identification of the single interchain disulfide bond (between Cys-80 in the a subunit and Cys-75 in the ,B subunit) of two-chain botrocetin confirmed that it is a heterodimer composed of a and 13 subunits and excluded the possibility of a mixture of two homodimers composed of a-a and 13-1 subunits. The similarity in location ofdisulfide bonds in these

Residue No.

Botrocetin a

p Echinoidin

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FIG. 5. Amino acid sequence homology between two-chain botrocetin and the known C-type lectins. Echinoidin, sea urchin lectin; HPCP, human proteoglycan core protein; HHLH2a, human hepatic lectin H2a; ABL, acorn barnacle lectin; RSL, rattlesnake lectin. Numbers indicate positions of amino acid residues from the N terminus. Gaps have been inserted to maximize homology. Identical residues to those of both the a and 3 subunits of two-chain botrocetin are boxed.

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C-type lectins is more obvious when compared with that of rattlesnake lectin recently published (26). Further studies on the structure-function relationship of the two forms of botrocetin should help clarify the mechanism of vWF-botrocetin complex formation, as well as the pathophysiological functions of vWF complexed to subendothelial components in the vessel wall. We thank Dr. Taei Matsui and Ms. Rieko Oyama for their excellent technical assistance and advice. We also thank Dr. Stephen Anderson for editing the manuscript. This work was supported in part by a Grant-in-Aid from the Fujita Health University (to K.T.), Grantsin-Aid for Scientific Research on Priority Areas (to K.T. and Y.F.), and Science Research Promotion Fund from Japan Private School Promotion Foundation (to K.T.). 1. Weiss, H. J. & Hoyer, L. W. (1973) Science 182, 1149-1151. 2. Zimmerman, T. S. & Meyer, D. (1987) in Haemostasis and Thrombosis, eds. Bloom, A. L. & Thomas, D. P. (Churchill Livingstone, London), pp. 131-147. 3. Sadler, J. E. (1991) J. Biol. Chem. 266, 22777-22780. 4. Howard, M. A. & Firkin, B. G. (1971) Thromb. Diath. Haemorrh. 26, 362-369. 5. De Marco, L. & Shapiro, S. (1981) J. Clin. Invest. 68, 321-328. 6. Read, M. S., Shermer, R. W. & Brinkhous, K. M. (1978) Proc. Nati. Acad. Sci. USA 75, 4514-4518. 7. Read, M. S., Potter, J. Y. & Brinkhous, K. M. (1983) J. Lab. Clin. Med. 101, 74-82. 8. Brinkhous, K. M., Read, M. S., Fricke, W. A. & Wagner, R. H. (1983) Proc. Nati. Acad. Sci. USA 80, 1463-1466. 9. Brinkhous, K. M., Smith, S. V. & Read, M. S. (1988) in Hemostasis and Animal Venoms, eds. Pirkle, H. & Markland, Jr., F. S. (Dekker, New York), pp. 377-398. 10. Brinkhous, K. M., Smith, S. V. & Read, M. S. (1991) in Handbook of Natural Toxins, ed. Anthony, T. T. (Dekker, New York), pp. 377-403. 11. Howard, M. A., Perkin, J., Salem, H. H. & Firkin, B. G. (1984) Br. J. Haematol. 57, 25-35. 12. Fujimura, Y., Holland, L. Z., Ruggeri, Z. M. & Zimmerman, T. S. (1987) Blood 70, 985-988. 13. Read, M. S., Smith, S. V., Lamb, M. A. & Brinkhous, K. M. (1989) Blood 74, 1031-1035. 14. Andrews, R. K., Booth, W. J., Gorman, J. J., Gastaldi, P. A. & Berndt, M. C. (1989) Biochemistry 28, 8317-8326.

Proc. Natl. Acad. Sci. USA 90 (1993) 15. Fujimura, Y., Titani, K., Usami, Y., Suzuki, M., Oyama, R., Matsui, T., Fukui, H., Sugimoto, M. & Ruggeri, Z. M. (1991) Biochemistry 30, 1957-1964. 16. Ruegg, U. T. & Rudinger, J. (1977) Methods Enzymol. 47, 111-116. 17. Hermodson, M. A., Ericsson, L. H., Neurath, H. & Walsh, K. A. (1973) Biochemistry 12, 3146-3153. 18. Masaki, T., Tanabe, M., Nakamura, K. & Soejima, M. (1981) Biochim. Biophys. Acta 660, 44-50. 19. Gross, E. (1967) Methods Enzymol. 11, 238-255. 20. Omenn, G. S., Fontana, A. & Anfinsen, C. B. (1970) J. Biol. Chem. 245, 1895-1902. 21. Mahoney, W. C. & Hermodson, M. A. (1980) J. Biol. Chem. 255, 11199-11203. 22. Knecht, R. & Chang, J.-Y. (1986) Anal. Chem. 58, 2375-2379. 23. Wilbur, W. J. & Lipman, D. J. (1983) Proc. Natl. Acad. Sci. USA 80, 726-730. 24. Smith, T. F., Waterman, M. S. (1981) Adv. Appl. Math. 2, 482-489. 25. Giga, Y., Ikai, A. & Takahashi, K. (1987) J. Biol. Chem. 262, 6197-6203. 26. Hirabayashi, J., Kusunoki, T. & Kasai, K. (1991) J. Biol. Chem. 266, 2320-2326. 27. Krusius, T., Gehlsen, K. R. & Ruoslahti, E. (1987) J. Biol. Chem. 262, 13120-13125. 28. Spiess, M. & Lodish, H. F. (1985) Proc. Natl. Acad. Sci. USA 82, 6465-6469. 29. Muramoto, K. & Kamiya, H. (1986) Biochim. Biophys. Acta 874, 285-295. 30. Fujimura, Y., Usami, Y., Titani, K., Niinomi, K., Nishio, K., Takase, T., Yoshioka, A. & Fukui, H. (1991) Blood 77, 113120. 31. Nishio, K., Fujimura, Y., Niinomi, K., Takahashi, Y., Yoshioka, A., Fukui, H., Usami, Y., Titani, K., Ruggeri, Z. M. & Zimmerman, T. S. (1990) Am. J. Hematol. 33, 261-266. 32. Fujimura, Y., Miyata, S., Nishida, S., Miura, S., Kaneda, M., Yoshioka, A., Fukui, H., Katayama, M., Tuddenham, E. G. D., Usami, Y. & Titani, K. (1992) Thromb. Haemostasis, 68, 464-469. 33. Mohri, H., Fujimura, Y., Shima, M., Yoshioka, A., Houghten, R. A., Ruggeri, Z. M. & Zimmerman, T. S. (1988) J. Biol. Chem. 263, 17901-17904. 34. Sugimoto, M., Mohri, H., McClintock, R. A. & Ruggeri, Z. M. (1991) J. Biol. Chem. 266, 18172-18178. 35. Drickamer, K. (1988) J. Biol. Chem. 263, 9557-9560.