Communication Vol. 266, No. 8, Issue of March 15, pp. 4665-4668,1991 THE JOURNAL OF BIOLOGICAL CHEMISTRY

0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U. S. A.

Self-association of Tissue Factor as Revealed by Chemical Cross- linking*

(Received for publication, November 12, 1990) Soumitra Roy, Lisa R. PaborskyS, and Gordon A. Vehars From the Department of Cardiovascular Research, Genentech Inc., South San Francisco, California 94080

The possible self-association of tissue factor mole- cules was investigated by treating cells expressing tis- sue factor with bifunctional cross-linking agents. The two reagents chosen were 3,3’-dithiobis(sulfosuc- cinimidylpropionate) and sulfosuccinimidyl 2-(p-azi- dosalicylamido)ethyl-1,3’-dithiopropionate, both of which are membrane-impermeable and thiol-cleava- ble. A human bladder carcinoma cell line, 582, and a transfected human kidney cell line expressing high amounts of recombinant tissue factor were used in these studies. Exposure of the intact cells to the cross- linking reagents was found to result in the formation of multimeric tissue factor-containing complexes, the extent of which appeared to be dependent upon the amount of tissue factor expressed by the cell. The self- association of tissue factor was prevented in a variant tissue factor molecule harboring a non-homologous transmembrane domain.

Tissue factor induces blood coagulation through its inter- action with plasma factor VII/VIIa on cell surface membranes (1). The membrane-bound complex of factor VII/VIIa with tissue factor is believed to activate the plasma proteins factor X and factor IX (2), which participate in the series of reactions leading to the formation of fibrin. Recent work on tissue factor protein characterization and cDNA cloning of its mRNA has revealed a protein component of approximately 30,000 daltons; three asparagine-linked carbohydrate struc- tures increase the apparent molecular weight, as determined by SDS-PAGE,’ to about 45,000 (1). Detailed structural in- formation and the availability of pure reagents have enabled detailed investigations into the structure/function aspects of this physiologically important coagulation cofactor,

Due to its key function in the initiation of blood coagula-

* 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Current address: Immulogic Pharmaceutical Corp., 855 California Ave., Palo Alto, CA 94304.

5 TO whom correspondence should be addressed Dept. of Cardio- vascular Research, Genentech Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080. Tel: 415-266-1084.

The abbreviations used are: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DTSSP, (3,3’-dithiobis(sulfo- succinimidylpropionate); SASD, sulfosuccinimidyl 2-(p-azidosalicy- lamido)ethyl-l,3’-dithiopropionate); S-2222, N-benzoyl-L-isoleucyl- L-glutamyl-glycyl-L-arginine-p-nitroanilide; PBS, phosphate-buff- ered saline; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfo~ic acid DTT, dithiothreitol; CI2Es, octaethyleneglycol dodecyl ether.

tion, several studies have investigated the interaction of factor VII/VIIa with tissue factor using a variety of normal and transformed cells that express tissue factor (3-6) as well as with purified tissue factor incorporated into phospholipid vesicles (7). It has been reported that factor VIIa binds to tissue factor with positive cooperativity; since the stoichiom- etry of binding of factor VII/VIIa to tissue factor was found to be 1:l at saturation (7), it was proposed that tissue factor exists as a dimer (7,8). We have investigated this hypothesis by using the membrane-impermeable, thiol-cleavable protein cross-linking agents DTSSP (9) and SASD (10). DTSSP has been used to investigate nearest neighbor relationships of the extracellular domains of integral membrane proteins (9, 11, 12). Both reactive moieties of DTSSP react with primary amine groups (on lysyl residues and available amino termini) whereas SASD has one amine-reactive and one non-selective, photoactivatable moiety. The present studies were performed using an immortalized human kidney cell line transfected with an expression plasmid directing the synthesis of tissue factor (13) as well as a human bladder carcinoma cell line, 582, which constitutively expresses high levels of tissue factor (6, 8). We show that tissue factor molecules can be cross- linked and that the cross-linking can be prevented by altering its transmembrane domain.

EXPERIMENTAL PROCEDURES

Materials-DTSSP and SASD were from Pierce Chemical Co. C,?Es was from Calbiochem. [35S]cysteine, [“Slmethionine, and [lZ5I] F(ab)z donkey anti-rabbit antibody were from Amersham Corp. Pro- tein G-Sepharose was from Pharmacia LKB Biotechnology Inc. DTT was from Bethesda Research Laboratories. Factor VI1 was from Sigma and factor X was from Enzyme Research Laboratories. The chromogenic substrate S-2222 for factor Xa was from Helena Labo- ratories. The rabbit antiserum RDO10, specific for human tissue factor, was made as described (14). Staphylococcus aurew protease V8 was from ICN. Purified Escherichia coli-expressed recombinant tissue factor truncated at residue 243 was supplied by Drs. Deborah Higgins and Tim Gregory (Genentech).

Vector Construction-The mammalian expression vector pCISTFl directing the synthesis of full-length tissue factor has been described (13). The mammalian expression plasmid pRK5 (15) was used to express two tissue factor variants. The EcoRI site located near the junction of the putative extracytoplasmic and transmembrane do- mains (13) was used for both constructs. The plasmid pRKTFdes243 was constructed by ligating a synthetic DNA fragment 5”AA TTC A G A G A A A T A T T C T A C A T C A T T G G A G C T G T G G T A T T T G T G G T C A T C A T C C T T G T C A T C A T C C T G G C T A T A T C T CTA CAC TAA (coding for the amino acid sequence EFREIFYII- GAVVFVVIILVIILAISLH stop) to the EcoRI site. This results in the synthesis of a tissue factor molecule with a truncation of the last 20 amino acids which constitute the cytoplasmic domain of tissue factor.’ The plasmid pRK5TFTM was similarly made by ligating the synthetic fragment 5“AA TTC AGA GAG CTC TTC ATA ATG A T C G T A G G A G G A C T A G T A G G C T T A A G A A T A G T T T T T GCA GTA CTA TCG ATA GTA CAC AAG TAA (coding for the amino acid sequence EFRELFIMIVGGLVGLRIVFAVLSIVHK stop). This results in the synthesis of a tissue factor variant in which the entire hydrophobic transmembrane domain has been replaced by a similarly hydrophobic transmembrane segment of the human im- munodeficiency virus envelope protein gp41 (16). The charged amino acids histidine (pRKTFdes243) or histidine and lysine (pRK5TFTM) have been retained to serve as membrane anchors. All constructions were verified by sequencing.

Cell Culture-The human bladder carcinoma 582 cell line

L. R. Paborsky and C. M. Gorman, unpublished data.

4665

4666 Chemical Cross-linking of Tissue Factor

(ATCC:HTB-1) was obtained from Dr. Walter Kisiel, University of New Mexico. The cells were maintained as described (6). The stable human kidney 293 cell clone transfected with the plasmid pCISTF1, 63.45.1, expressing human recombinant tissue factor was grown as previously described (13) in the presence of 50 nM methotrexate.

Transient transfections were done on 293 cells using the calcium phosphate precipitation method with 10 pg of DNA/lO-cm dish. Cells were used for cross-linking 14-20 h after transfection.

Cross-linking Conditions-Subconfluent cultures of the cells to be used for cross-linking were washed 3 times with cold endotoxin-free PBS or Hepes-buffered saline. The cells were resuspended in cold buffer (about 10 million cells/ml), and DTSSP was added to a final concentration as described in the figure legends. For cross-linking of adherent cells, DTSSP was added to washed cells on culture dishes. After cross-linking for 1 h on ice, the cells were washed 2 times with 50 mM arginine in PBS to remove and quench the cross-linker, and the cells lysed by incubating with extraction buffer (20 mM Tris-HCI, pH 7.5, 100 mM NaCI, 5 mM MgC12, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) on ice for 15 min, mixing intermit- tently. The extract was clarified by a 10-min centrifugation at 14,000 X g and then analyzed by SDS-PAGE and Western blotting using the polyclonal rabbit anti-tissue factor serum RDOlO (0.1 mg/ml) followed by '2sII-labeled anti-rabbit immunoglobulin G antibody (0.17 pCi/ml; 5 pCi/pg). Autoradiograms were scanned with an LKB 2202 Ultroscan laser densitometer equipped with a Hewlett-Packard 33904 integrator.

For cross-linking with SASD, the cells were washed with cold PBS as before and resuspended in 0.1 M sodium borate, pH 8.4, 0.05 M NaC1. A 5.5 mM SASD stock solution (0.11 M SASD in dimethyl sulfoxide diluted 20X in 0.1 M sodium phosphate, pH 7.4) was added to a final concentration as described in the figure legends. After 30 min at room temperature in the dark, 50 mM Tris-HCI, pH 8.4, was added as a free-radical scavenger (17) to minimize nonspecific photo- cross-linking. Cells to be cross-linked were exposed to long-wave ultraviolet light (365 nm) for 10 min in microtiter plate wells using the Stratalinker apparatus (Stratagene). Following cross-linking, tis- sue factor activity was assayed in the wells, or the cells were removed for analysis of cross-linking by Western blotting.

Tissue Factor Immunoprecipitation-Cells were labeled overnight with 100 pCi/ml of both [?3]cysteine and [%]methionine. Following cross-linking and extraction with extraction buffer, the extracts were incubated for 1 h with 3 pg of RDOlO followed by a 1-h incubation with Protein G-Sepharose. The immunoabsorbents were washed 5 times with RIPA buffer (1% Triton X-100,0.5% sodium deoxycholate, 0.1% SDS, 20 mM Tris-HCI, pH 7.5, 50 mM KCI, 1 mM EDTA) containing 0.5 M NaCl and 5 times with RIPA buffer. The Protein G-Sepharose beads were boiled in SDS-PAGE sample loading buffer to release the adsorbed proteins and the supernatant analyzed by 3- 10% gradient SDS-PAGE. For analysis of the gel slices under reducing conditions, the tissue factor aggregates were excised from the gel, soaked in 150 mM DTT overnight a t room temperature and re- electrophoresed on an 8% SDS-polyacrylamide gel. Peptide analysis of the '''S-labeled bands was carried out using S. aureus V8 protease exactly as described (18).

Tissue Factor Chromogenic Assay-This was carried out as de- scribed by O'Brien et al. (19). Briefly, to 50 pl of a cell suspension taken in a microtiter plate well were added 50 pl of 50 mM CaC12 and 50 pl of activation buffer (50 mM Tris-HC1, pH 8, 100 mM NaCI, 5 mg/ml bovine serum albumin, 0.1 units/ml factor VII, and 0.02 mg/ ml factor X, 10 mg/ml thimerosal). After 20 min a t room temperature, the factor Xa generated was quantitated by adding 50 pl of the chromogenic substrate S-2222 (reconstituted in water as instructed by the manufacturer). After 10-20 min at room temperature, 50 pl of 50% acetic acid was added to terminate the reaction and the absorb- ance at 405 nm recorded. Values used were the mean of assays carried out in triplicate.

RESULTS

Tissue factor being expressed on the surface of stably transfected 293 cells was found to be capable of being chem- ically cross-linked by the membrane-impermeable cross-link- ers DTSSP (Fig. 1, panel A ) and SASD (Fig. 1, panel B) . Western blot analysis of cell extracts following cross-linking revealed a pattern compatible with that expected from a multimerization of the 45-kDa tissue factor protein (bands corresponding to approximately 90,000, 135,000, 180,000, and

A B C D E

kpa 200 - 116-

68 - 97 - i 43 - I

"

-4 c

FIG. 1. Western blots showing cross-linking of tissue factor on cell surfaces. Panel A, cross-linking of tissue factor on clone 63.45.1 cells with (left to right) 0, 0.05, 0.1, 0.25, and 0.5 mM DTSSP in Hepes-buffered saline, respectively. Panel B, cross-linking of tissue factor on clone 63.45.1 cells with SASD; left to right, no SASD, UV alone, 0.16 mM, and 0.5 mM SASD, respectively. Panel C, cross- linking of tissue factor on clone 63.45.1 cells adherent to tissue culture plates. Left lane, PBS alone; right lane, 2.5 mM DTSSP in PBS. Panel D, cross-linking of tissue factor on the 582 bladder carcinoma cell line using (left to right) 0, 0.1, and 0.25 mM DTSSP. Panel E, cross- linking of purified recombinant tissue factor (cytoplasmic domain deleted; produced in E. coli; assembled into detergent C12Es micelles) using (left to right) 0, 0.1, and 0.25 mM DTSSP. The arrow indicates the position of the faster migrating band produced on cross-linking.

225,000 Da, respectively). A densitometric scan of a repre- sentative autoradiogam indicated that at least half of the detectable tissue factor was in the cross-linked aggregates (data not shown).

To address the possibility that the observed cross-linking was a result of the aggregation of cells in suspension, cross- linking was also carried out by incubating cells still adherent to tissue culture dishes with cross-linker, which produced a pattern similar to that seen using cells in suspension (Fig. 1,

Tissue factor expressed on the surface of the bladder car- cinoma cell line 582 could also be cross-linked (Fig. 1, panel D ) . This cell line expresses approximately 6 times less tissue factor than the transfected clone 63.45.1 (as determined by a tissue factor immunoassay of solubilized extracts). Although the higher multimers of tissue factor can be seen, the dimeric form predominates.

It has been shown that the tendency of purified tissue factor to aggregate can be greatly reduced by mutating the cyto- plasmic cysteine (13), and so it was of interest to determine whether purified recombinant tissue factor lacking the cyto- plasmic domain existed as aggregates in solution. Low con- centrations of cross-linker resulted in the production of a dimeric species and a faster migrating species probably re- sulting from an internal cross-link (Fig. 1,panel E, shown by an arrow).

To confirm that all the cross-linked molecules contained the 45-kDa tissue factor, the cells were metabolically labeled with [35S]cysteine and [35S]methionine and, cross-linking was carried out as before. Following lysis, tissue factor was ra- dioimmunoprecipitated, electrophoresed on a 3-10% gradient polyacrylamide gel, and the wet gel autoradiographed (Fig. 2). Gel slices corresponding to the visualized bands were cut out, reduced with dithiothreitol, and electrophoresed on an 8% polyacrylamide gel. Except for a 68-, a 29-, and a 14-kDa protein all the reduced gel slices yielded species which mi- grated at 45 kDa (Fig. 2).

That the higher multimers of tissue factor seen on cross- linking and immunoprecipitation contained only tissue factor moieties was confirmed by comparing the patterns produced by partial S. aureus protease V8 digestion (following reduction with 2-mercaptoethanol) of the 35S-labeled protein bands (Fig. 2) carried out as described (18). The 90-, 135-, and the 180- kDa bands produced a pattern identical to that produced by the 45-kDa tissue factor band. The patterns produced by digestion of the 29- and the 14-kDa bands are compatible

panel C ) .

Chemical Cross-linking of Tissue Factor 4667

97-

66’

43'

14-

234567

---

.

FIG. 2. Reduction with DTT and “Cleveland map” of tissue factor aggregates. Autoradiogram of’ a preparative :3-10?;, poly- acrylamide gel fbllowing cross-linking with IYI’SSI’ and electropho- rests of metabolically labeled clone 63.45.1 cells C/v/l ). Gel slices numbered l-7 as shown were reduced with I)TT and re-electropho- resed ([nncs l-7) on an 8% polyacrylamide gel (wntvr) along with “C-labeled molecular weight markers. The radiolabeled bands of the indicated molecular weights were also subjected “(‘leveland mapping” with S. (IUWU.S V8 protease and electrophoresed on a 15’; polyacryl- amide gel (r(qhi 1.

with the possibility of their arising from a proteolytic diges- tion of the 45.kDa tissue factor protein (20). The identity of the 6%kDa protein which was co-immunoprecipitated by the RDOlO antibody is unknown.

Because helix association of the transmembrane domains of receptor proteins has been implicated in the formation of oligomers (21), it was of interest to determine whether re- placement of the transmembrane region of tissue factor with that from a protein not known to be oligomeric would influ- ence cross-linking. A plasmid was constucted in which the tissue factor transmembrane amino acid sequence IFYIIGAV- VIILVIILAISL was replaced with the sequence LFI- MIVGGLVGLRIVFAVLSIV obtained from the human im- munodeficiency virus gp41 transmembrane domain and to which it bears no obvious similarity except for overall hydro- phobicity. This plasmid, pRK5TFTM, was transiently trans- fected into 293 cells along with another construct in which the native transmembrane sequence was retained (pRK5TFdes243). In both constructs the cytoplasmic domain has been deleted except for the charged amino acids histidine (pRK5TFdes243) or histidine and lysine (pRK5TFTM) im- mediately following the transmembrane domain, which were retained in order to serve as potential membrane anchors. Cross-linking of these cells (along with 63.45.1 cells) with DTSSP showed that whereas cells expressing tissue factor with only the cytoplasmic domain deleted could be cross- linked to dimers, the cross-linker has no discernible effect on tissue factor with the variant. transmembrane domain (Fig. 3). That the transfected cells were in fact expressing tissue factor correctly oriented on the surface was tested by using intact cells to activate factor VII in the tissue factor chro- mogenic assay. However, when the cells were lysed and the lysate tested for tissue factor activity following relipidation, tissue factor from pRK5TFTM-transfected cells was only about half as active in the chromogenic assay as tissue factor from pRK5TFdes243-transfected cells on a per molar basis (as estimated using a tissue factor immunoassay, data not shown). This loss of activity may be due to the loss of cooperativity in the binding of factor VII or more trivially to a difference in the efficiency of relipidation in the chromo- genic assay.

It was not possible to directly address the question of the relative fuctionalities of the cross-linked species because con- centrations of cross-linker which resulted in successful cross- linking also resulted in abrogation of tissue factor activity, as

FIG. :I. Western blot showing cross-linking of tissue factor variants. I,c’/f to ri,qh~. uncross-linked clone 63.45.1 cells: 63.4.5.1 cells cross-linked with 0.5 mM IYI’SSI’; 293 cells transiently trans- l’ected with the nlasmid ~)KK5TFdes243: r)HK5TFdes‘L43 transf’ected cells cross-linked with ti.5 mM I)TSSI’; 211:i cells transiently trans- I’ected with pRK5’I’FTM: J~HK~‘WI?VI transfected cells cross-linked with 0.5 mM D’I’SSI’.

did similar concentrations of the structurally similar lysine reactive reagent sulfosuccinimidobiotin (data not shown).

DISCUSSION

The process of blood coahwlation is localized to the site of injury as a result of the phospholipid dependence of the reactions that catalyze the generation of factor Xa and of thrombin. Each of these reactions has a requirement for a cofactor for the reaction to proceed: factor VIII for the acti- vation of factor X by factor IXa, factor V for the activation of prothrombin by factor Xa, and tissue factor for the acti- vation of factor X by factor VII(a). Similarity in protein function often can be correlated with similarity in structure. This is the case for factors V and VIII, two large, soluble plasma proteins; these two proteins have considerable se- quence identity and their proteolytic activation proceeds along very similar pathways (22-25). The resulting activated forms of these molecules consist of two (factor V) or three (factor VIII) nonconvalently associated subunits. These sub- units have been found to encode three homologous domains. By a mechanism that is not understood, this tri-domain interaction facilitates the activation of one vitamin K-de- pendent protease by a second vitamin K-dependent protease.

Tissue factor is able to accomplish an identical cofactor function in the activation of factor X by factor VII/VIIa. In striking contrast to the structure of factors V and VIII, however, the tissue factor protein is an integral membrane protein consisting of a short cytoplasmic domain, a transmem- brane domain, and a 220-residue external domain that does not share sequence identity with any known protein (1). Although t.issue factor might work through a fundamentally different cofactor mechanism than the other cofactors, we decided to investigate whether there may be an interaction of various tissue factor molecules that would in effect result in a multi-domain protein complex on the phospholipid surface and thereby tie its mechanism of act,ion to that of factors V and VIII. Such a possibility has been suggested by previous studies (7, 8) that suggested that tissue factor may function as a noncovalent dimer.

In this paper we have shown that tissue factor manifests a tendency to self-associate on cell surfaces as evidenced by successful chemical cross-linking. The multimeric complexes of 45.kDa units were shown to contain t.issue factor, and it is postulated that the pattern arises through the presence on the cell surface of some dimeric (and small amounts of higher multimeric) forms of tissue factor. The predominant species

4668 Chemical Cross-linking of Tissue Factor

seen on polyacrylamide gels was still the monomeric form. It is not known whether this pool of tissue factor unavailable for cross-linking was due to the fact that most of the tissue factor molecules were monomeric, or intracellular, or other- wise not accessible to cross-linker.

Appreciable amounts of complexes higher than dimers were seen by cross-linking tissue factor on the stable transfectant clone 63.45.1. Generation of complexes higher than dimer may have occurred by the incremental addition of monomer to the dimer. It is also possible that the SDS-PAGE pattern showing trimers, tetramers, etc. arose as a result of incomplete cross-linking within a larger complex. However, it is interest- ing to note that cross-links were never produced between tissue factor and other membrane proteins, as might be ex- pected to occur as a result of random cross-linking, as shown by the fact that all complexes produced the same “Cleveland map” as the monomeric tissue factor molecule. Moreover, the fact that we were able to inhibit self-association by changing the transmembrane domain is indicative of the fact that the cross-linking observed with the native molecule was specific.

Cross-linking with amino-reactive cross-linking reagents is dependent on the fortuitous presence of cross-linkable amino groups (a free NH2 terminus or lysine side. chains) within distances accessible to the cross-linker, 12 A in the case of DTSSP and 19 8, in the case of SASD. Thus, proteins which are known to interact cannot always be successfully cross- linked by chemical means. It was of obvious interest to determine whether factor VI1 could be included in a cross- linked complex with tissue factor on cell surfaces. Though factor VI1 could be shown to bind, we were unable to cross- link tissue factor to factor VI1 with either of these two reagents (data not shown).

Purified recombinant tissue factor produced in E. coli has been known to readily covalently aggregate in solution. This aggregation could be greatly reduced by mutating the cyto- plasmic’ cysteine to a serine (13). In mammalian cells, the cytoplasmic cysteine has been shown to be acylated (26) and is therefore presumably not available to form disulfide bridges. Therefore, deletion of the cytoplasmic domain would not be expected to affect cross-linking. This was confirmed both in uiuo (Fig. 3) and in vitro (Fig. 1, panel E ) . However, the formation of the internally cross-linked faster migrating spe- cies reducible with DTT (data not shown) observed on the addition of cross-linker to tissue factor in solution was never seen when tissue factor was cross-linked on the cell surface, suggesting that the membrane microenvironment is an im- portant determinant of tissue factor conformation. Replace- ment of the transmembrane region of tissue factor with an- other unrelated hydrophobic transmembrane segment was found to disturb the disposition of tissue factor molecules enough so as to prevent cross-linking when compared with molecules which possessed the correct sequence (Fig. 3).

While the transmembrane sequence appears to be necces- sary for interaction between tissue factor molecules, it re- mains to be determined whether it is sufficient. It is interest- ing to note that whereas the mouse and human tissue factor sequences are 60% identical, the analogy in the transmem- brane region is 48%, clustered in two short stretches (27). These are possible targets for site-directed mutagenesis ex-

periments to dissect out the precise residues potentially in- volved in the interaction.

The observation that the two membrane-impermeable amino-reactive cross-linkers tested in this paper both rapidly inactivated tissue factor may be useful in studies to investigate the phenomenon of tissue factor encryptation seen in various systems (1) whereby assayable tissue factor activity increases following cell lysis, and to determine whether this can be attributed to changes in the phospholipid environment. Cross- linking of 582 cells followed by lysis revealed little residual tissue factor activity (data not shown), confirming the pre- vious observation based on immunocytochemistry that tissue factor in these cells is almost entirely cell surface-expressed (28).

While cells in culture transfected and selected to produce very large amounts of protein is undoubtedly an artificial system, they serve as a useful system to monitor the properties of tissue factor. That tissue factor in 582 cells showed a similar cross-linking pattern to that of the transfected cells suggests that the observed multimerization represents a magnification of physiologically significant phenomena. Such systems there- fore should prove useful in resolving the control(s) in the function of this important component of the blood coagulation cascade.

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