0 1987 by The THE JOURNAL

Amencan Soclety for Bmchernistry OF B!OLOCIC?L CHEM!STRY

and Molecular Biology, Inc Vol. 262, No. 28, Issue of October 5, pp. 13835-13841,1987

Printed in U.S.A.

Isolation and Characterization of Two Domains of Human von Willebrand Factor That Interact with Fibrillar Collagen Types I and 111"

(Received for publication, June 4, 1987)

Francesco I. Pareti, Kenji Niiya, John M. McPhersonS, and Zaverio M. Ruggerii From the Scripps Clinic and Research Foundation, La Jolln, California 92037 and the $Collagen Corporation, Palo Alto, California 94303

We have identified four discrete proteolytic frag- ments of von Willebrand factor (vWF) that define two collagen-binding domains. Two of the fragments tested, T 96 kDa and T 55 kDa, were generated by digestion with trypsin, and two, Fragments I and 111, with Staphylococcus aureus VS protease. The larger Fragment 111, a disulfide-linked homodimer, extends between residues 1 and 1365 of the 2050-residue vWF subunit and comprises the sequence of all the others. T 96 kDa, also a disulfide-linked homodimer, extends between residues 449 and 728. T 55 kDa and Fragment I, both single-chain polypeptides, have a partial se- quence overlap corresponding to residues 91 1-1 114, and together extend from residue 730 to 1365. The ability of the fragments to interfere with the vWF- collagen interaction was evaluated by measuring in- hibition of '261-labeled vWF binding to fibrillar bovine collagen types I and 111. All the four fragments tested inhibited binding. Native conformation was essential for expression of this function; denaturation with gua- nidine hydrochloride and reduction of disulfide bonds resulted in marked reduction or complete loss, respec- tively, of the inhibitory activity at all the coocentra- tions tested. Two monoclonal antibodies were pre- pared, one directed against T 96 kDa and the other against Fragment I. Both antibodies partially inhibited vWF binding to collagen, and their inhibitory effect was enhanced when they were used together. I2'I-La- beled Fragment I bound to collagen in a saturable manner, and the binding was completely blocked by both T 96 kDa and T 55 kDa. Thus, we have identified at least two distinct functional domains of vWF that concurrently mediate the vWF-collagen interaction. The two domains appear to share a common recogni- tion site on collagen.

von Willebrand factor (vWF)' plays an essential role in primary hemostasis by anchoring platelets at the site of

* This work was supported in part by Grants HL 31950 and HL 37522 from the National Institutes of Health. This is Manuscript 4860 BCR from the Division of Experimental Hemostasis, Depart- ment of Basic and Clinical Research, Research Institute of Scripps Clinic. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

and Research Found., 10666 N. Torrey Pines Rd., La Jolla, CA 92037. J To whom reprint requests should be addressed Scripps Clinic

I The abbreviations used are: VW, von Willebrand factor; HPLC, high-performance liquid chromatography; SDS, sodium dodecyl sul- fate.

vascular injury (1-3). This function is expressed through the interaction with platelet receptors (4) as well as collagen (5- 20) and noncollagenous components of the subendothelium (21, 22). The latter may include sulfated glycolipids (23, 24) and/or heparin-like molecules (25, 26).

Studies over the past several years have provided informa- tion on the interaction between vWF and collagen. Early reports established that vWF binds equally well to the differ- ent types of genetically distinct interstitial collagens (8, 9) and that optimal binding occurs when tropocollagen is orga- nized into polymeric structures (10, 11). In fact, monomeric collagen inhibits binding to fibrils only when present at high concentration; heat-denatured collagen has no inhibitory ef- fect (10, 11). Native conformation of vWF appears to be important for the interaction since limited reduction of mul- timeric vWF to its dimeric form was found to result in markedly decreased binding to collagen, and complete reduc- tion to the single-chain constituent subunit completely abol- ished binding (11, 18).

More recent studies, however, have provided results in apparent contrast with those reported previously. We have found that a reduced and S-carboxymethylated tryptic frag- ment of vWF, extending between residues Val449 and Lys7" of the constituent subunit, completely blocks the binding of 'Y-labeled vWF to acid-insoluble, covalently cross-linked equine collagen type I (19). Moreover, other investigators (20) have described two denatured fragments of vWF, generated by treatment of the reduced and S-carboxymethylated protein with cyanogen bromide, that retain the ability to interact with monomeric type I11 bovine collagen. One of the two fragments extends between residues 542 and 662 of the vWF subunit, in agreement with our previous findings (19), and the other between residues 948 and 998.

The present series of experiments was performed with the purpose of characterizing the vWF domains that bind to fibrillar collagen types I and 111. The results obtained dem- onstrate the existence of at least two collagen-binding do- mains in human vWF that appear to interact with the same site(s) on collagen, as judged by cross-inhibition studies. The native conformation of these vWF domains appears essential for their function, and optimal binding of vWF appears to require the concurrent interaction of both domains with col- lagen.

EXPERIMENTAL PROCEDURES

Purification and Characterization of u WF-vWF was purified and characterized as previously described (4, 27). Starting material was cryoprecipitate of normal plasma (a generous gift of American Red Cross) for the vWF employed in binding studies and commercial factor VI11 concentrates (a generous gift of Armour) for experiments in which the protein was subjected to proteolytic fragmentation (27).

13835

13836 von Wilkbrand Factor-Collagen Interaction Generation and Characterization of Proteolytic Fragments of v WF-

Purified vWF, at a concentration of 2-5 mg/ml in a buffer composed of 0.05 M Tris-HC1 and 0.15 M NaC1, pH 7.4, was digested with Staphylococcus auwus V8 protease following the procedure described by Girma et al. (28) with minor modifications. The amount of enzyme used was '/40th that of substrate on a molar basis. After a 48-h incubation at 27 "C, the digestion was terminated by addition of 2 mM diisopropyl fluorophosphate. The mixture was dialyzed against 50 mM Tris-HC1, pH 7.4, and the proteolytic fragments were sepa- rated into three fractions (Fragments 1-111, respectively, based on the order of elution) by means of ion-exchange chromatography on a Mono-Q column (Pharmacia P-L Biochemicals) using a 0-1 M linear gradient of NaCl. This was followed by gel filtration on a Sephadex G-75 column (Pharmacia P-L Biochemicals) in order to remove S. aureus V8 protease. The cumulative yield of the three fragments was greater than 80% of the amount of vWF digested. Fragment 111 was further purified by affinity chromatography on heparin-Sepharose (Pharmacia P-L Biochemicals) after dialysis against 0.2 M ammonium acetate (25). Fragment 111, which contains two heparin-binding do- mains (25, 26), bound to the column and was eluted with a 0-1 M linear gradient of NaC1; contaminating Fragment I1 did not bind to heparin.

Digestion of vWF with trypsin and separation of the resulting fragments into several fractions using gel filtration were performed as previously reported (27). In preliminary experiments, we observed that fractions B and C inhibited binding of lZ5I-labeled vWF to bovine collagen types I and 111 (see below). Therefore, the main component of each fraction was purified by ion-exchange high-performance liquid chromatography (HPLC; Waters) using a Mono-& column (Phar- macia P-L Biochemicals) eluted with a 0-0.5 M linear gradient of NaCl in 50 mM Tris-HC1 buffer, pH 7.8. Two fragments were ob- tained. One, of apparently 45 kDa unreduced and 55 kDa after reduction of disulfide bonds, was eluted at NaCl concentrations between 0.14 and 0.16 M; the other, of apparently 96 kDa unreduced and 52/48 kDa after reduction, was eluted at 0.33-0.35 M NaCl. In subsequent experiments, trypsin-digested vWF was subjected directly to ion-exchange chromatography for initial separation of the two fragments of interest. Final purification of the two fragments was achieved by HPLC molecular sieve chromatography using two Zorbax columns (Du Pont-New England Nuclear), one GF-450 and one GF- 250, mounted in series and eluted with 0.4 M ammonium phosphate, pH 7. The final yield of these two purified fragments was between 5 and 10% (weight-to-weight) of the amount of vWF digested. After purification, all the proteins and fragments were dialyzed against a buffer composed of 0.02 M Tris-HCI, 0.15. M NaC1, pH 7.4 (Tris- buffered saline), and stored at -70 "C until used. Purified fragments were characterized by polyacrylamide gel electrophoresis and immu- noblotting, both performed following publihhed techniques (29-31). Reduction and.S-carboxymethylation of proteins as well as amino- terminal sequence analysis were performed as reported (26). The carboxyl-terminal sequence of the trypsin-generated polypeptide of 55 kDa (see below) was determined as previously described for the 52/48-kDa fragment of vWF (26) (kindly performed by Dr. Koiti Titani, Fujita-Gakuen Health University, Toyoake, Japan).

Production and Characterization of Monoclonal Antibodies-Two murine monoclonal anti-vWF antibodies, identified as RG46 and FrI- 3, were prepared as described previously (31) using as immunogens reduced vWF subunit and intact S. aureus V8 protease-generated Fragment I, respectively. The antibodies were characterized using a microtiter assay with insolubilized antigen in native form or by immunoblotting of SDS-denatured, unreduced or reduced antigen. Monoclonal IgGs were purified from ascites fluid using either DEAE- Affi-Gel Blue (Bio-Rad) or Protein A-Sepharose (Sigma), as previ- ously described (32,33), and stored after dialysis against Tris-buffered saline. Immunoblotting was performed after transfer of antigen onto nitrocellulose membranes, as described (34).

Binding of uWF and Fragment I to Collagen-The ligands used in these experiments were radiolabeled with '''I following the technique described by Fraker and Speck (35) using IODO-GEN (Pierce Chem- ical Co.). The radiolabeled '251-labeled vWF retained its native mul- timeric structure (4). Specific activity of the radiolabeled ligands was between 2.46 and 8.55 X Ci/mg (or 9.13 and 31.7 X lo6 Bq/mg). Intact bovine collagens, types I and 111, were purified from calf skin as previously described (36, 37). Intact type I and 111 collagen fibrils were prepared from a solution of monomeric collagen under low ionic strength conditions at 15 "C as previously described (38), and the suspensions (1-3 mg/ml) were stored at 4 "C until used. Ten to twenty rl of the suspension (depending on the desired final collagen concen-

tration) and 10 ~1 of a buffer composed of 0.02 M mono- and disodium phosphate and 0.13 M NaCl, pH 7.4 (phosphate-buffered saline), were used in each binding mixture along with the other reagents, as described below. A commercial preparation of pepsin-solubilized bo- vine dermal collagen (Vitrogen 100, Collagen Corp.) was used in some experiments instead of the intact type I collagen and gave identical results. In this case, fibril formation was initiated by adjusting the pH to 7.4 using %oth-final volume of a buffer containing 0.2 M mono- and disodium phosphate and 1.3 M NaC1, pH 7.4, and '/loth-final volume of 0.1 M NaOH. The collagen solution was rapidly diluted to 1.3 mg/ml using phosphate-buffered saline and then 20 p1 were pipetted into conical polystyrene tubes and incubated at 37 "C for 30 min. At this point, the other components of the binding mixtures, including radiolabeled ligand, proteolytic fragments tested for their inhibitory activity, or Tris-buffered saline, were added to give a final volume of 75 pl. The mixtures, all prepared in duplicate or triplicate, were immediately mixed and then incubated for 20 min at room temperature (22-25 "C). Preliminary experiments demonstrated that the binding was independent of temperature in the range of 4-37 "C and that equilibrium was reached in 20 min (19). Separation of hound from free ligand was obtained by centrifugation at 12,000 X g for 8 min in a microcentrifuge (Beckman) at room temperature.The super- natant containing free ligand was carefully removed, and the tube tips containing collagen fibrils and bound ligand were cut and counted in a y-scintillation spectrometer (Packard Instrument Co.). Negative controls consisted of mixtures in which phosphate-buffered saline was substituted for collagen. Nonspecific (nonsaturable) binding was evaluated in each experiment by adding 100-fold excess of unlabeled ligand; in some cases, it was calculated from total binding isotherms using the computer-assisted program LIGAND (39). The multimeric structure of bound vWF was analyzed by SDS-agarose electrophoresis (40).

Inhibition binding studies were performed using various proteolytic fragments of vWF or purified IgG of the monoclonal antibodies. The proteolytic fragments were added to the experimental mixture im- mediately before addition of the radiolabeled vWF, whereas the monoclonal antibodies were incubated with vWF for 10 min at room temperature before addition to the other components of the experi- mental mixture. The assay was then performed as described above. Binding measured in the presence of the inhibitor tested was ex- pressed as percentage of that measured in a control mixture in the absence of inhibitor after subtraction from each value of the binding measured in the presence of 100-fold excess of unlabeled ligand (nonspecific binding).

Measurement of Protein Concentration-The concentration of monoclonal IgG was calculated from absorbance at 280 nm using an extinction coefficient of 1.43 (1 mg/ml concentration). The concen- tration of vWF and its fragments was also calculated from absorbance at 280 nm with correction for light scattering measured at 320 nm. The calculated extinction coefficient (1 mg/ml) was 0.8. The values estimated by this method were in close agreement to those obtained with the method of Hartree (41) using bovine serum albumin as standard.

RESULTS

Characterization of the Proteolytic Fragments of u WF-Two S. aureus V8 protease-generated fragments, Fragments I and 111, and two trypsin-generated fragments, T 96 kDa and T 55 kDa (identified by their apparent molecular mass), were pu- rified under native conditions. Their electrophoretic mobility in SDS and their reactivity in immunoblotting are shown in Figs. 1 and 2, and a schematic representation of their location in the vWF subunit is shown in Fig. 3. Fragment I, which did not react with any of the available monoclonal antibodies after exposure to SDS, was identified by its electrophoretic mobility and by amino-terminal sequence analysis, both cor- responding to those previously reported by Girma et al. (28) and Titani et al. (42), respectively. The amino-terminal se- quence and carboxyl-terminal residue of the T 52/48-kDa fragment, extending between residues Val4*' and Lys728 and representing the constituent subunit of the dimeric T 96-kDa fragment, as well as the amino-terminal sequence of the T 55-kDa fragment have been reported in previous publications (19, 26, 27). The carboxyl-terminal residue of the T 55-kDa

uon Willebrand Factor-Collagen Interaction 13837

M l M19 M31

225 koa- 175 koa-

110 koa-

45 koa-

Coomassie Autoradiography

- - "- A vWF F r I F r l I Fr lU F r l I F r I I I F r l I F rm F r I I F r m

FIG. 1. Characterization of the proteolytic fragments of vWF generated by digestion with S. aureus VS protease. The panel on the left shows the three purified fragments (Fr I , Fr I I , and Fr I I I ) and the vWF used as substrate for the digestion ( vWF) . Proteins were stained with Coomassie Blue after electrophoresis performed in a 6.5% polyacrylamide gel with 0.174% bisacrylamide in the presence of SDS according to the method of Laemmli (29). All samples were analyzed after reduction of disulfide bonds with 10 mM dithiothreitol. The three panels on the right show the results of the immunoblotting of Fragments I1 and 111. Following polyacrylamide gel electrophoresis, the proteins were transferred onto nitrocellulose (34) and incubated with specific anti-vWF mouse monoclonal anti- bodies that had previously been shown to react with known regions of the vWF subunit (31), as indicated by the corresponding cyanogen bromide fragments (M) numbered according to Titani et al. (42). Positive reactivity was demonstrated by incubation with '2sII-labeled rabbit anti-mouse IgG followed by autoradiography. M7 extends between residues 185 and 288 of the vWF subunit, M19 between residues 844 and 947, and M31 between residues 1481 and 1693 (42). Note that Fragment 111 (residues 1-1365) reacts with antibodies against M7 and M19, but not M31; Fragment I1 (residues 1366-2050) reacts only with antibodies against M31. The calculated apparent molecular mass of the vWF subunit and the purified fragments (after reduction of disulfide bonds) is indicated on the left. The minor bands seen in the vWF, Fr II, and Fr III lanes are due to uncontrolled proteolysis of the purified vWF preparation (see Ref. 31).

fragment was tentatively located a t Arg"I4, thus establishing an overlap (from Gly9'' to Arg1'I4) between Fragment I and T 55 kDa (Fig. 3).

Binding of 'ZsI-Labeled u W F to Bovine Collagen Types I and III-Two experiments were performed with each type of collagen using different preparations of radiolabeled vWF. The results, summarized in Table I, demonstrate that more vWF bound, on a weight-to-weight basis, to collagen type I11 than to type I. The affinity constant, calculated with the computer-assisted program LIGAND (39), was greater for collagen type I than for type 111, whereas the calculated nonsaturable binding was very similar in both cases. Analysis of bound vWF by SDS-agarose electrophoresis demonstrated that all multimeric forms interacted with collagen, but a relatively greater proportion of larger multimers was bound to collagen type I as compared to type I11 (Fig. 4).

Effect of Different Proteolytic Fragments on the Binding of '"I-Labeled u W F to Collagen-All the proteolytic fragments were initially tested after purification under native conditions. The 5'. aureus V8 protease-generated Fragments I11 and I both inhibited the binding of 12sI-labeled vWF to bovine collagen types I and 111, but Fragment I was less effective than Fragment I11 (Fig. 5). The concentration of Fragment I11 necessary to inhibit 50% of vWF binding (ICso) was between 0.36 and 0.66 p~ with collagen type I (range of three experiments) and between 0.73 and 1.1 p~ with collagen type I11 (range of two experiments). The ICso values for Fragment I were between 1.4 and 1.5 p~ with collagen type I (range of three experiments) and 2.8 p~ with collagen type I11 (one

96kDa-

Coomassie Autoradiography Coomassie Autoradiography

52148kDa- - 55kDa

NR R NR R R R FIG. 2. Characterization of the proteolytic fragments of

vWF generated by digestion with trypsin. The purified trypsin- generated fragments were analyzed by electrophoresis in a 10% pol- yacrylamide gel containing 0.174% bisacrylamide and in the presence of SDS (29). The samples were tested either unreduced ( N R ) or after reduction of disulfide bonds with 10 mM dithiothreitol ( R ) . The two panels on the left show the 96-kDa trypsin-generated fragment (ap- parent molecular mass with intact disulfide bonds) visualized by Coomassie Blue staining (Coomassie) or by immunoblotting (Auto- racliography) with a monoclonal anti-vWF antibody reacting with cyanogen bromide fragment M9 (residues 293-540 of the vWF sub- unit), as described in the legend to Fig. 1. Note that after reduction, a closely spaced doublet of 52/48 kDa is seen; this heterogeneity in molecular mass is the consequence of different glycosylation, as previously discussed (27). The panel on the right shows a similar analysis of the 55-kDa trypsin-generated fragment. In this case, electrophoresis was performed after reduction of disulfide bonds ( R ) using a 15% polyacrylamide gel, and immunoblotting was performed with a monoclonal antibody reacting with cyanogen bromide fragment M19 (residues 844-947 of the vWF subunit). The apparent molecular mass of the T 55-kDa fragment with intact disulfide bonds is 45 kDa (not shown here). See the legend to Fig. 1 for additional technical details.

1 vWF Constituent Subunit 2050

H,N &' & COOH

1 1365

6 I

(;I" SVa Fragment JI ldimerl

0 911 1365

b 449

SVs Fragment I

I

I T 96 kDa (dimer of T 52148 kDal

730

b-; 1 5 5 koa

FIG. 3. Schematic representation of the vWF subunit and its proteolytic fragments. One-letter abbreviations are used to indicate amino acids at the amino and carboxyl termini of the vWF subunit and of the fragments generated by S. aureus V8 protease (SV8) and trypsin (T). The vWF subunit is represented in its reduced state (single-chain polypeptide), whereas the proteolytic fragments are represented as obtained from digestion of vWF under nonreducing conditions. Thus, S. aureus V8 protease-generated Fragment 111 (28) and T 96 kDa are homodimeric molecules, whereas S. aureus V8 protease-generated Fragment I (28) and T 55 kDa are single-chain molecules.

experiment). After exposure to the denaturing agent guani- dine HCl or reduction and alkylation of disulfide bonds, the ability of Fragment I11 to inhibit vWF binding to fibrillar bovine collagen types I and I11 was markedly reduced or completely lost, respectively (Fig. 6). Fragment I was largely insoluble after reduction and alkylation and could not be tested under these conditions.

We also evaluated the inhibitory effect of the trypsin- generated fragments, T 96 kDa and T 55 kDa. In a previous publication (19), we showed that the reduced and alkylated polypeptide extending from Val4"' to Lys7", the subunit con-

13838 von Willebrand Factor-Collagen Interaction TABLE I

Binding of 1251-labeled uon Willebrand factor to collagen Fibrillar collagen type I (pepsin-digested) or I11 was used in these

experiments at a final concentration of 310 or 533 pg/ml, respectively. 12sI-Labeled vWF was used at concentrations between 0.78 and 200 pg/ml. Analysis of binding isotherms was performed using the com- puter-assisted program LIGAND (39), and nonsaturable (nonspecific) binding was calculated as a fitted parameter. Nonsaturable binding was also evaluated experimentally using a 100-fold excess of unlabeled vWF relative to the concentration of '251-labeled vWF used. Because of limitations in the concentration of available vWF, this could only be done when the labeled ligand was present a t 20 pg/ml or less. The calculated and experimentally determined valued were in close agree- ment.

Collagen type '9 -vWF bound Km Nonsaturable binding

pmol u WF subunitlmg collagen M' Bovine type I 5.11 4.23 X lo7 3.43 X 10-2

6.99 8.12 X 107 3.44 X 10-2

Bovine type I11 40 1.86 X 107 5.55 X 10-2

61 0.96 X lo7 2.69 X

0 1 2 3

FIG. 4. Analysis of the multimeric structure of vWF bound to collagen. This analysis was performed after separating bound from free ligand as described under "Experimental Procedures." Col- lagen with bound vWF was solubilized in SDS-containing buffer, and the samples were analyzed by agarose electrophoresis, as described (40). The gel was then fixed and dried, and an autoradiograph was obtained. Samples were applied at the top of the gel (cathode). The distribution of vWF multimers is a function of their size, with smaller multimers moving progressively toward the anode (bottom). In this gel system, each multimer appears as three bands (indicated by the brace). Lane I, the radiolabeled vWF used as ligand (41 ng); lane 2, the vWF bound to collagen type I (49 ng); lune 3, the vWF bound to collagen type I11 (52 ng). The inset shows a laser densitometer scanning of the three lanes, demonstrating the different relative proportion of multimeric forms.

stituent of the dimeric T 96-kDa fragment (Fig. 3), completely blocked vWF binding to the equine collagen used in that study; whereas the T 55-kDa peptide (fraction C ) had only a partial inhibitory effect and only when used at higher concen- trations. In these studies, we found that both fragments, when maintained under native conditions, blocked vWF binding to fibrillar bovine collagen types I and 111, and T 55 kDa was at least as effective as T 96 kDa or better (Fig. 7). The inhibitory effect observed with T 55 kDa corresponded, on a molar basis, to that seen with Fragment I (compare Figs. 5 and 7). Thus, inhibition of vWF binding to type I11 collagen required more competing fragment than inhibition of binding to type I collagen, a likely consequence of the greater amounts of vWF bound to collagen type 111. In contrast to the effect on binding to equine cross-linked collagen type I (19), neither trypsin- generated fragment showed any significant inhibitory effect after reduction and alkylation (Fig. 7).

100 -

.- P 80- -U r

"""

.- m

6 0 - 5 -

LC) N c - m 40 a R

-

CT

v) W

* 20 -

O0.01 0.1 1 10

Added Fragment (pM1 FIG. 5. Inhibition of vWF binding to different types of col-

lagen by S. aureus VS protease generated Fragments I and 111. Fibrillar collagen was used at final concentrations of 350 pg/ml (type I, pepsin-digested) and 270 pg/ml (type 1111, and radiolabeled vWF was at a final concentration of 2 pg/ml. Since the amount of bound vWF varied with the different type of collagen used (see Table I), the specific binding measured in a mixture containing Tris- buffered saline instead of competing fragments was considered as loo%, and the residual binding measured in the presence of varying amounts of Fragment I or I11 was calculated as a percentage of that. Nonsaturable binding was estimated in the presence of a 100-fold excess of unlabeled vWF in the control mixture and was subtracted from total binding measured for each experimental point. See "Ex- perimental Procedures" for additional technical details.

SV8-Generated Fragment III

= 100- m E?l Bovine Collagen Type I c

c .- $ 0 Bovine Collagen Type m - K 50 - - - m =I

v) (u

-cl

cc n- s Reduced and Denatured Native

FIG. 6. Effect of denaturation of Fragment I11 on the inhi- bition of vWF binding to collagen. The Fragment 111 used in this experiment was either maintained under nondenaturing conditions or denatured by dialysis against 7 M guanidine HCI with or without subsequent reduction and alkylation, as indicated. Both reduced and unreduced preparations were extensively dialyzed against Tris-buff- ered saline.before testing. The experimental conditions were as de- scribed in the legend to Fig. 5. Note that denatured, reduced and alkylated Fragment 111 did not significantly inhibit vWF binding to collagen types I and 111 even though it was used at concentrations (10 p ~ ) in excess of those necessary to block completely vWF binding when the fragment was maintained in native conditions (see Fig. 5).

Alkylated (Guanidine-HCI)

Binding of '251-Labeled Fragment I to Different Types of Bovine Collagen-Fragment I bound in a saturable manner to fibrillar bovine collagen types I and I11 (Table 11). At variance with the results obtained using "'I-labeled vWF, the amount of Fragment I bound at saturation and the affinity constant were very similar with both collagen types I and 111. The affinity constant of '2sI-labeled Fragment I for collagen was much lower than that of intact vWF and compatible with the

values obtained from inhibition of vWF binding by un-

von Willebrand Factor-Collagen Interaction

T 96 kDa (pM) T 55 kDa (pM1 .- F l o o r n T

l o o r 0.5

50 n t Native (Oimert

100 r 0.5

n Native (Dimer)

Bovine Collagen Type I

R-A Native R-A (Monomert (Single Chainl

Bovine Collagen Type llI

R.A lMonomerl

Native R.A (Single Chainl

FIG. 7. Inhibition of vWF binding to collagen by two tryp- sin-generated fragments. The binding of '251-labeled vWF to bo- vine fibrillar collagen types I (pepsin-digested) and 111 was measured in the presence of each fragment at the concentrations indicated for each column. The results are expressed as percentage residual binding (specific) relative to a control mixture containing Tris-buffered saline instead of fragment. The two trypsin-generated fragments (T frag- ments) are identified with their apparent molecular masses. The T 96-kDa fragment is a homodimer (each constituent chain has an apparent molecular mass of 52/48 kDa), and the T 55-kDa fragment is a single-chain molecule. Note that neither fragment retains inhib- itory activity after reduction and alkylation (R-A) . See the legend to Fig. 5 for additional technical details.

TABLE I1 Binding of '251-labeled Fragment I to collagen

In these experiments, 1251-labeled Fragment I was used at concen- trations between 3.1 and 400 pg/ml. Binding was measured and analvzed using the procedure described for Table I.

1261-Labeled

bound Collagen type Fragment I K . Nonsaturable

binding

pmollmg collagen " Bovine type I 101 8.91 X lo6 1.57 X lo-'

162 5.05 X lo5 1.63 X lo-' Bovine type 111 103 4.05 x 10' 1.63 x lo-'

193 4.31 X lo5 1.57 X lo-'

labeled Fragment I (see above). The binding of '251-labeled Fragment I to collagen was blocked by native vWF as well as by both the tryptic fragments, T 96 kDa and T 55 kDa, that inhibited the binding of intact vWF to the same types of collagen (Fig. 8).

Effect of Monoclonal Antibodies on the Binding of lZ5I- Labeled uWF and 1251-Labeled Fragment I to Collugen-One of the two anti-vWF monoclonal antibodies selected for this study, RG46, reacted with intact vWF, the trypsin-generated T 96-kDa homodimer, and with its constituent chain, the T 52/48-kDa fragment obtained after reduction and alkylation (Fig. 3). The other antibody, FrI-3, reacted only with native

-6 Buffel

13839

n r vWF T96kDa T55kDa

FIG. 8. Effect of two trypsin-generated fragments on the binding of Fragment I to collagen. The binding of '251-labeled Fragment I (added at a final concentration of 30 pg/ml) to bovine fibrillar collagen type I (pepsin-digested) was measured as described under "Experimental Procedures." The inhibitory effect of the T 96- and T 55-kDa fragments (see legend to Fig. 7) as well as of intact vWF was measured at the micromolar concentrations indicated for each column. The specific binding (total minus nonsaturable) meas- ured in a mixture containing Tris-buffered saline instead of compet- ing ligand was taken as loo%, and the results are expressed as a percentage of that binding. Nonsaturable binding was that measured in the presence of a 100-fold excess of unlabeled Fragment I. Note that both fragments inhibit the binding of Fragment I to collagen, although only T 55 kDa has a partial sequence overlap with it.

TABLE 111 Inhibitory effect of monoclonal antibodies against T 96 kDa and Fragment I on 1251-labeled u WF binding to bovine collagen type I Fibrillar collagen type I (pepsin-digested) was used at a concentra-

tion of 310 pglml, and 1251-labeled vWF was used at 2 pg/ml. Binding in the presence of the indicated concentrations of purified monoclonal anti-vWF IgG is expressed as percentage of that measured in a control mixture tested at the same time and containing the same concentra- tions of an irrelevant monoclonal IgG (anti-thyroglobulin). "'I-La- beled vWF was incubated with monoclonal IgG for 10 min at room temperature before addition of the collagen.

Antibody Concentration Residual vWF binding

mglml

0.12 0.04 0.01

0.12 0.04 0.01

Anti-T 96 kDa 0.5

Anti-Fragment I 0.5

Anti-T 96 kDa + anti- 0.12 + 0.12 Fragment I

% of control 43 37 60

102 57 53 78 96 19

Fragment I and vWF. Both antibodies inhibited '"I-labeled vWF binding to fibrillar collagen type I, although only par- tially (Table 111). When used together, the two antibodies had a greater inhibitory effect than when used singly (Table 111). Antibody FrI-3 (anti-Fragment I) completely inhibited the binding of 1z51-labeled Fragment I to fibrillar collagen type I, whereas RG46 (anti-96-kDa fragment) was without effect in this regard, thus suggesting that the two antibodies recognize different epitopes on the two distinct proteolytic fragments of vWF.

DISCUSSION

In this report, we characterize four discrete proteolytic fragments of human vWF that inhibit binding of the intact

13840 uon Willebrand Factor-Collagen Interaction

molecule to fibrillar collagen types I and 111. One of these fragments, T 96 kDa, is a dimer whose constituent subunit extends between residues 449 and 728 of the 2050-residue vWF subunit. Two other fragments, T 55 kDa and S. aureus V8 protease-generated Fragment I, are single-chain polypep- tides that have a sequence overlap corresponding to residues 911-1114, and together represent the subunit sequence be- tween residues 730 and 1365. The larger of the fragments described here, S. aweus V8 protease-generated Fragment 111, is a homodimer of a chain extending between residues 1 and 1365 and includes the sequence of the other three fragments (Fig. 3). We assume that these proteolytic fragments of vWF define at least two distinct collagen-binding domains, one corresponding to the T 96-kDa polypeptide and the other to the overlap region between T 55 kDa and Fragment I (Fig. 3).

A previous report (11) has suggested that collagen quater- nary structure, rather than collagen type per se, is the main factor determining the affinity of vWF binding. Consequently, for the present series of experiments, we elected to measure vWF binding using suspensions of fibrillar collagen. More- over, our choice is justified by the consideration that in the vessel wall, where the vWF-collagen interaction is likely to play a physiological role, collagen types I and I11 are present as organized fibers. We found that binding to collagen type I was less than to type 111. A reasonable explanation for this observation resides in the different geometry of the fibrils formed since these are much thicker with collagen type I than with type I11 and offer less surface available for interaction with macromolecular ligands (8). The quantitatively re- stricted binding of vWF to collagen type I resulted in prefer- ential binding of the larger multimeric forms (Fig. 4), a finding that explains the greater association constant measured with collagen type I as compared to type 111 (Table I). In fact, vWF molecules with higher degree of polymerization bear a greater number of binding sites and, as expected for polyvalent li- gands, express higher affinity. Thus, under conditions in which a limited number of binding sites are available on collagen, larger multimers bind in relatively greater propor- tion. The concept that the molecular size of the ligand may limit the binding to the thick fibrils formed by collagen type I is supported by the results obtained with Fragment I; this single-chain polypeptide of approximately 45 kDa, as opposed to up to lo4 kDa for the multimeric vWF (43), bound in equal amounts and with similar association constant to both colla- gen types I and 111.

Although we used suspensions of fibrillar collagen, our estimates of maximal vWF binding per unit weight of collagen and association constants are in reasonable agreement with those reported by investigators who used monomeric collagen insolubilized onto plastic surfaces. The values we obtained are closer to those of Bockenstedt et al. (17) than to those of Roth et al. (20). Barring experimental variability, the greater binding reported in the latter study may be due to coating of the plastic surface with significantly less collagen (up to 1000 times less) than used by Bockenstedt et al. In fact, steric hindrance of large vWF multimers bound may prevent the binding of additional molecules to surrounding areas, thus resulting in an estimated lower binding to surfaces coated at a higher density. Differences in the affinity constants reported in these studies are possibly due to different multimeric composition of the labeled vWF used according to the reasons explained above.

Our present results demonstrate that under the conditions used in this study, expression of function in the isolated proteolytic fragments defining the collagen-binding domains of vWF requires maintenance of native conformation. The S.

aureus V8 protease-generated Fragment I and T 55 kDa can be isolated as single-chain polypeptides under nonreducing conditions and contain 4 and 18 cysteine residues, respectively (42); T 96 kDa is a disulfide-linked homodimer. Reduction of one or more crucial disulfide bond(s) or alteration of disulfide- determined three-dimensional structure within these regions of vWF appears to impair severely the interaction with col- lagen, as measured by blocking of intact vWF binding. In accordance with these findings, other investigators (11, 18) previously reported that reduction of vWF adversely affected its ability to bind to collagen.

Their results and our present findings, however, are in apparent contrast with those recently obtained by Roth et al. (20) who found that two reduced and alkylated cyanogen bromide fragments of vWF, M11 and M20, could interfere with binding of the intact molecule to monomeric type I11 collagen. Neither of these fragments contains cysteine resi- dues (42). It appears therefore that the primary structure of these two regions of the vWF subunit contains the informa- tion sufficient for recognition of specific interaction sites on collagen, although expression of the binding function in the intact molecule or even smaller proteolytic fragments requires an appropriate three-dimensional conformation dependent on secondary and tertiary structure. In fact, the isolated vWF fragments containing the functional domains described in this report exhibited less activity even after denaturation without reduction of disulfide bonds. Our results nevertheless concur with those of Roth et al. in identifying the regions of vWF involved in the interaction with collagen. In fact, the T 96- kDa fragment, a dimer of the T 52/48-kDa polypeptide that extends between residues 449 and 728 of the vWF subunit, comprises the sequence of cyanogen bromide fragment M11 (residues 542-622), whereas the sequence common to the two polypeptides, T 55 kDa and S. aureus V8 protease-generated Fragment I (residues 911-1114), includes that of cyanogen bromide fragment M20 (residues 948-998).

We have previously characterized the interaction of vWF with fibrillar type I collagen prepared from horse tendon and obtained results that differ from the present ones in two respects. First, in those experiments (19), binding was inhib- ited effectively by the T 96-kDa dimer (contained in fraction B described in that study) but also by its purified, reduced and alkylated constituent chain, the 52/48-kDa polypeptide. Second, the T 55-kDa fragment (contained in fraction C described in that study) had only limited inhibitory activity and only at the higher concentrations tested. In experiments not reported here, these differences were confirmed using all the vWF fragments described in this study. In view of the conserved nature of the collagen molecule, it would not be anticipated that the corresponding binding domains of vWF could exhibit such significant functional differences based solely on the different origin of the collagen used equine in the previous study (19) and bovine in this one. A possible explanation might be found in the different physicochemical state of the collagen preparations since the equine collagen previously used was chemically cross-linked to render it acid- insoluble (19). This raises the possibility that in physiological conditions, the interaction of collagen with other molecules in the subendothelium, for example heparin-like glycosami- noglycans, may in turn modulate the vWF-collagen interac- tion. Additional work is required to clarify these points.

Roth et al. (20) have suggested that the two collagen-binding domains of vWF may represent a duplication of the same functional site since they are encoded in repeat regions of the vWF gene and contain homologous amino acid sequences. In accordance with this concept, we demonstrate here that the

uon Willebrand Factor

T 96-kDa peptide inhibits the interaction of Fragment I with collagen, a result providing evidence that these two domains, although apparently distinct, may share a common interaction site on collagen. Nevertheless, the concurrent function of both domains is necessary for an optimal interaction between vWF and collagen. In fact, blocking of either functional domain with specific monoclonal antibodies resulted in a marked, although only partial, reduction in vWF binding to collagen; a greater inhibitory effect was observed when two antibodies directed against distinct epitopes located in the 52/48-kDa domain or Fragment I domain were used in combination.

In conclusion, the isolation and characterization of proteo- lytic fragments of vWF containing the collagen-binding do- mains of this molecule provide the background information for future work aimed at the definition of the essential struc- tural features that support this function.

Acknowledgments-We wish to thank Paul Kostel and Susan Rus- sell for expert technical assistance and Claire Jackson and Kay Baylor for excellent secretarial assistance.

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