The EMBO Journal vol.4 no.12 pp.3153-3157, 1985

Molecular cloning of S-, a link between complement, coagulation and cell-substrate adhesion

Dieter Jenne' and Keith K.Stanley2 bystander cells against lysis by fluid phase C5b-7 and the inhibi- lInstitute of Medical Microbiology, Justus-Liebig-University in Giessen, tion of C9 polymerisation during fluid phase assembly. Schubertstrasse 1, 6300 Giessen, and 2European Molecular Biology S-protein may also have a physiological role in the coagulation Laboratory, Meyerhofstrasse 1, Postfach 10.2209, 6900 Heidelberg, FRG pathway since S-protein can be observed in a complex with Communicated by R.Cortese thrombin in serum (after coagulation), but not in plasma (Podack and Muller-Eberhard, 1979). This complex has been shown to cDNA clones coding for human S-protein have been isolated be a stable trimolecular complex containing antithrombin HI in using monoclonal antibodies to screen a cDNA library in pEX. addition (Jenne et al., 1985a). S-protein can modulate the ac- These clones are shown to be authentic S-protein clones on tivity of thrombin by annulling the heparin-dependent activation the basis of sequence, composition and immunological criteria. ofthe thrombin inhibitor, antithrombin Il (Preissner et al., 1985), The complete open reading frame sequence for S-protein has and by a direct reduction of antithrombin IH inhibition of throm- been determined and shows it to be a single polypeptide chain bin (Jenne et al., 1985a). of 459 amino acids preceded by a cleaved leader of Here we report the molecular cloning and cDNA sequence of 19 residues. No evidence was found for polymorphism of S- S-protein. Comparison of this sequence with partial peptide se- protein suggesting that different molecular weight forms arise quence data of the serum spreading factor called '' by proteolytic degradation. Of the first 44 amino-terminal (Holmes, 1967; Hayman et al., 1982, 1983; Barnes and Silnutzer, residues 42 are identical with the so-called somatomedin B 1983) shows that the two are identical. peptide suggesting that S-protein is the somatomedin B precursor. Striking homology is found in the rest of the se- quence with the serum spreading factor, vitronectin, which Results has also been shown to contain somatomedin B sequences at Identification and sequencing of S-protein cDNA its amino terminus. We conclude that S-protein and vitronec- A mixture of five monoclonal antibodies raised against S-protein tin are identical and discuss the relevance of this finding to purified from SC5b-9 complexes (Jenne et al., 1985b) was used the coagulation and complement pathways. to screen a subcloned human liver cDNA expression library in Key words: cell spreading/SC5b-9 complex/somatomedin B/ the bacterial expression vector, pEX (Stanley and Luzio, 1984). thrombin/vitronectin From 30 000 independent clones in the library we obtained five clones which expressed antigenic determinants of S-protein (Figure 1). These were colony purified and found to contain Introduction cDNA inserts which cross-hybridised. All the clones contained S-protein is found at high concentrations in human plasma large open reading frames assessed by the size of the hybrid pro- (140-700 jig/ml, Podack and Miiller-Eberhard, 1979; Jenne et teins on Western blots showing that the S-protein antigenic deter- al., 1985b; Dahlback and Podack, 1985) and is able to bind to minants were unlikely to be expressed from cDNA fragments protein complexes in the terminal stages of both the complement fused to the expression vector in missense reading frames. The and coagulation pathways. Stable complexes of S-protein with the terminal complement components have been observed after (a) Si C5 is activated via the alternative pathway (Kolb and Muller- S2 Eberhard, 1975; Bhakdi and Roth, 1981), in C8- or C9-depleted S3/S4/S5 serum (Podack et al., 1977; Bhakdi and Roth, 1981), and with S108 detergent-solubilised C5b-9 (Bhakdi and Tranum-Jensen, 1982b). S203 In all these situations the C5b-7 complex is generated in the S5.1 absence of a target lipid bilayer. Binding of S-protein to this fluid SS2 phase C5b-7 may prevent its attachment to cell membranes, and (b) Apal rStuI) Apal Pvun NaeI ApaI although C8 and a few molecules of C9 can still bind (Kolb and the terminal complex contain- lc) 4w 4w *. -*. -4- cytolytic ----0- Miiller-Eberhard, 1975), -4- ing polymerised C9 is not formed (Podack et al., 1984; Dahlback -4- and Podack, 1985). SC5b-9 complex formation has also been ----w inferred during complement attack on some bacteria (Joiner et al., 1982) and in systemic lupus erythematosus (Falk et al., 0 500 1000 1500 1985). If the S-protein is dissociated from the fluid phase com- Fig. 1. S-protein cDNA clones. (a) Clones SI to S5 were obtained by plex by detergent or proteolytic treatment apolar surfaces become expression screening of a human liver cDNA library in pEX. Clones S108 and the resulting complexes aggregate (Bhakdi et al., and S203 were obtained by DNA hybnrdisation screening of a parent library exposed in pKT218. (b) Shows the assembled structure of the S-protein cDNA with 1979; Podack and Miller-Eberhard, 1980; Bhakdi and Tranum- restriction enzyme sites as described in the text. The open box represents Jensen, 1982a). The functions of S-protein may therefore be the the open reading frame. (c) Shows the overlapping M13 clones used to solubilisation of fluid phase C5b-9 complexes, the protection of sequence the cDNA.

(©) IRL Press Limited, Oxford, England 3153 D.Jenne and K.K.Stanley

150 M AP LORP L L IL ALL -15

200 A W V A L A D _Q E S C K G R C T E G F N V D K K C Q C D E L C S V V (a)D 0 E S C K G R C T E G F N V D K K C Q C D K L C S V V 1 10 20 201 ACCAGAGCTGCTGCACAGACTATACGGCTGAGTGCAAGCCCCAAGTGACTCGCGGGGATGTGTTCACTATGCCGGAGGATGAGTACACGGTCTATGACGA 300 Q C C T D Y T A E C K P Q V T R G D V F T M P E D E Y T V S D D Q 5 N C T C V T A E C K P Q V T (b)P E L E V T V Y D S 30 40 50 60 301 TGGCGAGGAGAAAAACAATGCCACTGTCCATGAACAGGTGGGGGGCCCCTCCCTGACCTCTGACCTCCAGGCCCAGTCCAAAGGGAATCCTGAGCAGACA 400 G EE K N N A T V R EQ V G G P S L T S D L 0 A QOS K G N P EQ T G EE K N S AT V ? Q V G 70 80 90 401 CTTCGACTAGAAGCCGGCGGTGCCTTACTAGGTGCCAGCGGCCTACAGAA 500 P V L K P EKEE A P AP E V G AS K P EGSI DO 0 P ET L N P D R P 100 110 120 501 O P P A KEE EL COD K PPF D A FT D L K N GOSL F A FORG 0 V C 130 140 150 160 601 CTATGAACTGGACGAAAAGGCAGTGAGGCCTGGGTACCCCAAGCTCATCCGAGATGTCTGGGGCATCGAGGGCCCCATCGATGCCGCCTTCACCCGCATC 700 Y E L D E K A V R P G V P K L I R D V W G ISE G PSI D A A FT MOI 170 180 190 701 800 N C Q G K TVY L F K DRN 0 V W R F ED G V L D P D V PMR NI S D G F (c)P 0 V P C F I S C G F 200 210 220 801 900 D D IP DRN V D A A LA L P A RHS V S G RE R V VPF F K G K Q V W ?FOIP ? N V? A 230 240 250 260 GGAGTACCAGTTCCAGCACCAGCCCAGTCAGGAGGAGTGTGAAGGCAGCTCCCTGTCGGCTGTGTTT'GAACACTTTGCCATGATGCAGCGGGACAGCTGG 1000 K Y Q F 0 NOQ P S Q E EC E GOSS L S A V F ENH PAN NONQ D S W 270 280 290 1001 EDOI F E LL F W D R TOS A G TOG P Q FISKR D W N G V P G Q V D 300 310 320 1101 1200 A A MADG ROIY IOSG M A PR P S L T K K Q N FM NORN OK GYMR (d)A P 0 P 5 L A K K Q R F 0 N 0 N R K G V 0 330 340 350 360 1201 1300 O ORG NO RDG R N O N 0MRM P0 A_M_W L S L P S S E E S N L G SOQRD NOSR D R NOQN 0MM P S 370 380 390 1301 GCAACAGTATCGAGATGTGGCGCCTTACCTCGGGCTTCTTTGGCATCACA 1400 A N N V D DVY RNM D W L V P AT C K P0I00 V F F FOSG D K Y Y R V 400 410 420 1401 N LORT R R V D T V D P P V P RO I A Q V W L G C P AP GOH 440 450 1501 GCGGCAAGCGGCTTTGTCTCCCTTCTCCACCAAAGCCTGCCAAAAAAAAA 1600 1601 AAAA 1604 Fig. 2. The nucleotide and amino acid sequence of S-protein. Random fragments of clones S108 and the ends of S203 were sequenced in M13 and assembled by computer. The deduced amino acid sequence is shown in single letter code beneath the DNA sequence together with the sequence of fragments of the proteins somatomedin B (a) and vitronectin (b - d). Fragments (b) and (d) were generated by cyanogen bromide cleavage while fragment (c) was cleaved using acid (Suzuki et al., 1984). Underlined asparagine residues are possible sites for attachment of N-linked oligosaccharide, and the dashed lines show the amino-terminal sequences of S-protein fragments previously determined (Dahlback and Podack, 1985). clone with the longest cDNA insert, S3, was chosen as a DNA Figure 2. probe to screen the parent cDNA library in pKT218 (Woods et The cDNA sequence of S-protein is 1604 bp long including al., 1982) by hybridisation. The 13 clones obtained in this way a 61-bp untranslated region at the 5' end and a stretch of 109 were analysed by digestion with Pvull and Pstl and then by bases containing the polyadenylation signal AAUAAA at the 3' hybridisation with a PvuH-BamHl fragment derived from the 5' end. The open reading frame codes for 478 residues, of which end of S3. The clone having the longest fragment at the 5' end, the first 19 appear to be a cleaved leader peptide since they are S 108, was sequenced on both DNA strands in M13 by the very hydrophobic in character, and end with an alanine residue shotgun procedure of Sanger et al. (1977). This cDNA clone which is a suitable cleavage site for signal peptidase. was also capable of expressing S-protein antigenic determninants The predicted amino acid composition of the mature protein when subcloned into pEX (see Figure 3), showing that the an- agrees well with that determined experimentally in several dif- tigenic determinants of S-protein were not present at the boun- ferent laboratories (Table I). In addition the amino termiinal dipep- dary between the fl-galactosidase of the expression vector and tide of the mature protein agrees with the published the expressed cDNA fragment. In order to obtain the 5' end of amino-terminal sequence of S-protein (Dahliback and Podack, the S protein cDNA it was necessary to performn a second screen- 1985). In order to distinguish different epitopes encoded by the ing of the library in pKT2 18 using the PstI/StuI fragment from cDNA clones we subcloned the two halves of clone S5 digested the 5' end of S108 as a probe. Among 12 clones isolated, one, with NaeI into pEX and tested the individual expressed fragments S203, was estimated to be 100 bp bigger than S108 by restric- with the monoclonal antibodies. The amino-terminal portion of tion enzyme mapping. The additional sequence data from this this clone bound monoclonal antibodies 1-4, while the carboxy- clone completed the coding sequence of the S-protein shown in terminal hybrid protein bound only monoclonal antibody 5 3154

A Molecular cloning of S-protein

Table I. Amino acid composition of S-protein and vitronectin 1 2 3 4 5 Amino acid (1) (2) (3) (4) (5) (6) r--- S 11 Ala 29.8 31.9 28.0 29.8 30.8 28 Cys 9.6 15.0 9.6 11.2 9.0 14 Asp 47.3 52.4 55.5 43.5 50.5 S2 Asn 16 34 tv- Glu 62.9 67.0 61.5 62.9 66.1 Gin 26 S3 A-- iOi Phe 24.3 25.9 23.9 22.5 22.9 23 39.0 37 Gly 38.1 42.6 32.1 37.5 - Wi His 9.6 10.5 10.1 17.2 9.3 9 __i Ile 13.3 15.5 11.9 14.2 13.0 13 54 Lys 22.0 27.4 27.1 19.5 21.8 20 Leu 26.2 30.7 37.6 30.0 28.1 25 Z Met 8.3 5.8 7.8 6.7 5.1 7 Pro 35.8 39.1 29.8 33.0 38.5 35 Arg 36.7 37.4 27.1 33.7 34.7 35 - - u io_. @ i 33 ..eivi- Ser 30.8 38.2 34.9 33.0 28.0 S10855- Thr 19.3 20.9 24.8 18.7 17.4 19 Val 22.0 28.1 23.0 21.0 23.5 21 6.0 n.d. 9 Trp 4.1 6.0 n.d. : H:t 'a ZuS >e i... 23 Ie Tyr 22.5 23.0 14.2 21.0 21.4 S521 f Comparison of the amino acid composition calculated on the basis of a weight of 52 371 daltons for S-protein (1 -4), vitronectin (5) and molecular to expressed fragments of S-protein the cDNA sequence (6). Values are from (1) Podack and Mulier-Eberhard Fig. 3. Monoclonal antibody binding (4) cDNA. cDNA clones obtained from the human liver library in pEX or (1979), (2) Dahlback and Podack (1985), (e) Jenne et al. (1985b), in Figure 1. and (5) Barnes and Silnutzer (1983). subloned into pEX from a library in pKT218 were as shown Preissner et al., (1985) after restriction enzyme n.d., not determined. SS.1 and S5.2 refer to the two halves of S5 cleavage at the NaeI site and subcloning of the fragments into pEX. (Figure 3). The epitope for this antibody is therefore located acids of the protein. 1), we were unable to detect any differences in the restriction within the last 84 amino digestion with ApaI Since the cDNA clones express at least two distinct epitopes fragment lengths of S-protein cDNA after well with the published which cuts the S-protein cDNA at the extreme ends of the open of S-protein, and the sequence agrees of S3-5 is probably amino terminal sequence and composition, we conclude that this reading frame (Figure 1). The extra length for authentic S-protein. due simply to longer poly(A) tails. sequence codes polypeptide estimated from the cDNA S-protein The size of the S-protein Proteolytic degradation of sequence is 52 371 daltons as compared with reported values bet- Purified S-protein separates into two major bands on SDS poly- ween 66 and 89 kd according to the method used (Podack and acrylamide gels with apparent mol. wts. of - 65 and 75 (Podack Muller-Eberhard, 1979). This difference is presumably due to and Muller-Eberhard, 1979; Preissner et al., 1985; Dahlback the addition of carbohydrate to the molecule and possibly also and Podack, 1985; Jenne et al., 1985b). In addition a small pep- due to an unusual secondary structure since S-protein has a high tide of - 12 kd has been reported in SDS gels of reduced prepara- proportion of proline residues. Three possible attachment sites tions of purified S-protein (Podack and Muller-Eberhard, 1979; for N-linked oligosaccharide are found in the sequence (underlin- Dahlback and Podack, 1985) and in plasma where it has been ed in Figure 1). positive (Jenne et al., 1985b; Preissner et shown to be immune of S-protein with vitronectin al., 1985). Since this fragment is only released after reduction, Comparison it is most likely Comparison of the S-protein sequence with the protein sequence and it can react with S-protein-specific antibodies, Foundation derived from the intact molecule by . S-protein data library of the National Biomedical Research gives rise to a similar pattern of showed a close homology of S-protein with somatomedin B digested in vitro with trypsin other significant bands on SDS gels (Podack and Muller-Eberhard, 1979). A possi- (Fryklund and Sievertsson, 1978). No the se- homologies were detected. Part of the amino-terminal sequence ble location for a cleavage point is at residue 380, since serum-spreading quence at this position has been reported as a contaminating of somatomedin B has also been reported in the and Podack, factor called vitronectin (Suzuki et al., 1984). We therefore com- amino-terminal sequence of S-protein (Dahlback protein with 1985). The fragment released by cleavage at this position would pared the partial amino acid sequence data for this it can be seen that the that of the S-protein (Figure 1). In addition to the somatomedin have a mol. wt. of 9.4 kd. In Figure 3 molecule 3' end of clone S5 digested with NaeI expresses the epitope of B sequence all other sequenced regions of the vitronectin NaeI cuts the S-protein cDNA such showed a very similar and sometimes identical sequence. In each monoclonal antibody 5. Since agreed that only four amino acids in addition to the carboxyterminal frag- case the amino acid residues preceding these sequences likely that endogenous proteases with their origin as cyanogen bromide or acid-cleaved fragments. ment are expressed, it is very factor can cleave S-protein at or near to this site. When the amino acid composition of the serum-spreading origin of heterogeneity is differential splic- was compared with that of the S-protein, we found a close cor- Another possible proteins are ing within the S-protein mRNA. Although clones S3, S4 and S5 respondence (Table I), also suggesting that the two did appear to have slightly longer 3'-PvuH-PstI fragments (Figure very similar, if not identical.

3155 D.Jenne and K.K.Stanley

Discussion domain is the tripeptide Arg-Gly-Asp which is shared by several We have described cDNA clones coding for the entire open molecules having cell attachment activity (Hayman et al., 1985; reading frame of S-protein. These cDNA molecules code for at Pierschbacher and Ruoslahti, 1984). Somatomedin B isolated least two distinct epitopes recognised by a total of five monoclonal from plasma is sometimes found with the Arg of this tripeptide antibodies. Since the antigen used to raise these monoclonal anti- at its carboxy terminus (L.Fryklund, personal communication) bodies was S-protein dissociated from SC5b-9 complexes, and suggesting that it might be the product of S-protein after inac- the same monoclonal antibodies label S-protein complexed to tivation of its spreading activity. It will be interesting to deter- thrombin-antithrombin III from serum (Jenne et al., 1985b), it mine if thrombin binding or cleavage occurs at this site since is beyond reasonable doubt that these cDNA clones code for thrombin cleaves the same dipeptide (Arg-Gly) in fibrinogen. authentic S-protein. In addition, the amino terminal dipeptide and Previous experiments investigating cleavage of S-protein or composition deduced from the cDNA sequence closely agree with vitronectin by thrombin could only show a cleavage at the car- the data obtained from S-protein purified from plasma in several boxytenninal end of the molecule which removes both the heparin different laboratories (Podack and Muller-Eberhard, 1979; -binding site [see (iv) below] and the trypsin fragment [see (v) Dahlback and Podack, 1985; Jenne et al., 1985b; Preissner et below; Podack and Miuller-Eberhard, 1979; Silnutzer and Barnes, al., 1985). 1984]. (iii) There then follows a long region (residues 48- 347) The sequence of S-protein shows an unexpected homology with which contains the three possible sites for attachment of N-linked two other related plasma proteins, somatomedin B and vitronec- oligosaccharides. This region is rich in proline residues but shows tin. Somatomedin B was originally characterised as a growth- no amino acid homology with collagen or fibronectin. It is also hormone-dependent polypeptide with mitogenic activity towards densely scattered with hydrophobic residues. (iv) The following human glial cells (Uthne, 1973) but later its mitogenic activity 32 residues of the molecule contain 14 positive charges, no was accounted for by small contaminations with EGF (Heldin negative charges, and only two hydrophobic residues. This region et al., 1981). In this respect it no longer qualifies for the term has been identified in vitronectin as a heparin-binding site (Suzuki 'somatomedin'. Of the first 44 amino acids of S-protein, 42 are et al., 1984). The ability of S-protein to abolish the heparin identical to those of the somatomedin B peptide (Fryklund and stimulation of antithrombin 11 inactivation of thrombin (Preissner Sievertsson, 1978). The two differences are most likely the result et al., 1985) is likely to be due to a direct interaction of heparin of difficulties in the amino acid sequencing (L.Fryklund, per- with S-protein at this site. This site could also be involved in sonal communication). S-protein is therefore presumably the the binding of S-protein to the terminal components of the com- precursor of somatomedin B, although no independent function plement pathway since it has been observed that poly-lysine can has yet been ascribed to the released peptide. block the inhibition of C5b-7 by serum factors (Lint et al., 1976). Vitronectin (so called because of its affinity for glass surfaces) (v) Immediately following the heparin-binding region is a site is a cell spreading factor from serum (Holmes, 1967; Barnes et at which S-protein is found to be partially cleaved in plasma and al., 1980; Hayman et al., 1982) which has been shown to con- which is rapidly cleaved when the purified protein is treated with tain the first 30 residues of somatomedin B at its amino terminus trypsin (Podack and Miller-Eberhard, 1979). Endogenous proteo- (Suzuki et al., 1984) and which is functionally but not immuno- lytic cleavage of this carboxy-terminal fragment accounts for the logically related to fibronectin (Barnes et al., 1980; Hayman et two molecular weight forms of both S-protein and vitronectin al., 1983). Comparison of the sequence, biochemical and (Suzuki et al., 1984). Using five different monoclonal antibodies biophysical properties shows that S-protein and vitronectin are no evidence was found for multiple forms of S-protein in the indistinguishable. In addition to the 30 amino acid residues of cDNA library. Although not all clones were sequenced, the in- somatomedin B at the amino terminus of vitronectin, three pep- ternal restriction fragments from all the clones were indistinguish- tide fragments within the molecule have 68 out of 76 residues able suggesting that only one S-protein polypeptide is synthesised in common with S-protein. Furthermore the amino acid com- in human liver. This does not rule out the possibilty that a dif- position of serum-spreading factor determined by a different ferent type of vitronectin is made in tissues other than liver. laboratory is very similar to that of S-protein (Barnes and The identity of S-protein and vitronectin leads to new concepts Silnutzer, 1983; Table I). Both proteins are present at a similar in the control of the complement and coagulation pathways. concentration in plasma (Barnes et al., 1983; Podack and Muller- Fundamental to these is the observation that S-protein (as Eberhard, 1979; Jenne et al., 1985b), have a similar multiple- vitronectin) is present not only in plasma, but also on the sur- banded appearance on SDS polyacrylamide gels (Suzuki et al., face of many cells, in loose connective tissue (Hayman et al., 1984; Hayman et al., 1983; Podack and Miuller-Eberhard, 1979; 1983) and in platelets (Barnes et al., 1983). In this respect it is Dahlback and Podack, 1985; Jenne et al., 1985b), and adhere like fibronectin, although the two molecules are distinct and even strongly to surfaces (Holmes, 1967; Podack and Miller- where they are co-localised they are present at different concen- Eberhard, 1979). We conclude therefore that the two molecules trations. When the endothelial cells lining blood vessels are are identical. damaged, fibronectin-like molecules (fibronectin and von Wille- Using the combined information about S-protein and vitronectin brand factor) mediate the adhesion of platelets thus localising the we are able to divide the S-protein sequence into five regions deposition of platelets and fibrin in the vicinity of the lesion with different functional significance. (i) The first 44 residues (Sixma and Wester, 1977; Giddings, 1983). During this process of S-protein (somatomedin B) contain four disulphide bonds the transglutaminase activity of factor XIIIa covalently cross-links (Fryklund et al., 1974) and has the appearance of an independent- fibronectin to fibrin and collagen. If S-protein were also exposed ly folding cysteine-rich domain. No homology was observed bet- at these sites it would be expected that it could play an important ween this region and the high cysteine regions of complement role in the localisation of the terminal components of both the component C9, factor IX or wheat germ agglutinin which repre- complement and coagulation pathways at the site of injury. Clot- sent classes of conserved high cysteine structural motifs found ting could be potentiated both by the local retention of thrombin in several other plasma and membrane proteins (Stanley et al., to the exposed sub-endothelial matrix and by its protection against 1985a, 1985b). (ii) Immediately following the somatomedin B inactivation by antithrombin III. In a similar way S-protein could z 3156 Molecular cloning of S-protein contribute to the local assembly of terminal complement com- Podack,E.R. and Muller-Eberhard,H.J. (1980) J. Immunol., 124, 1779-1783. ponents or the removal of fluid phase complexes from the cir- Podack,E.R., Kolb,W.P. and Miiller-Eberhard,H.J. (1977) J. Immunol., 119, 2024-2029. culation under pathological conditions. This prediction is Podack,E.R., Preissner,K.T. and Muller-Eberhard,H.J. (1984) Acta Pathol. supported by the observation that terminal complement com- Microbiol. Immunol. Scand. Ser. C, Suppl. 284, 92, 89-96. ponents are deposited at atherosclerotic plaques (Niculescu et al., Preissner,K.T., Wassmuth,R. and Muller-Berghaus,G. (1985) Biochem, J., 231, 1985; De Heer et al., 1985) and also by the striking co-localisa- 349-355. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA, tion of vitronectin and terminal complement complexes in kidney 74, 5463-5467. tissue (Hayman et al., 1983; Falk et al., 1983). Silnutzer,J. and Barnes,D.W. (1984) Biochem. Biophys. Res. Commun., 118, Vitronectin is a member of a family of substrate adhesion 339-343. molecules which include collagen, fibronectin, chondronectin, Sixma,J.J. and Wester,J. (1977) Semin. Hematol., 14, 265. laminin, thrombospondin and von Willebrand factor (Hynes and Stanley,K.K. (1983) Nucleic Acids Res., 11, 40774092. Stanley,K.K. and Luzio,J.P. (1984) EMBO J., 3, 1429-1434. Yamada, 1982; Edelman, 1985). It has not until now, however, Stanley,K.K., Kocher,H.-P., Luzio,J.P., Jackson,P. and Tschopp,J. (1985a) been ascribed a unique function. We have shown that plasma EMBO J., 4, 375-382. vitronectin is the same protein as S-protein suggesting that it is Stanley,K.K., Page,M., Campbell,A.K. and Luzio,J.P. (1985b) Mol. Immunol., a multifunctional protein that can interact with both the terminal in press. Suzuki,S., Pierschbacher,M.D., Hayman,E.G., Nguyen,K., Ohgren,Y. and components of the complement and coagulation pathways and Ruoslahti,E. (1984) J. Biol. Chem., 259, 15307-15314. also with cells and matrix constituents. Uthne,K. (1973) Acta Endocrinol. Suppl., 175, 1-26. Woods,D.E., Markham,A.F., Ricker,A.T., Goldberger,G. and Colten,H.R. Materials and methods (1982) Proc. Natl. Acad. Sci. USA, 79, 5661-5665. AU methods relating to the cloning and expression of S-protein were as previously Received on 2 September 1985 described (Stanley, 1983; Stanley and Luzio, 1984). Acknowledgements Note added in proof D.J. would like to thank Profs. S.Bhakdi and H.-J. Wellensiek for their support While this manuscript was in press the sequence of vitronectin cDNA has been and encouragement during this work. published (Suzuki,S., Oldberg,A., Hayman,E.G., Pierschbacher,M.D. and Ruoslahti,E. (1985) EMBO J., 4, 2519-2524). This sequence is included within the S-protein sequence starting at base 71 and is identical except for three omis- References sions (at positions 541, 551, 561) and four substitutions (at positions 736, 1157, Bhakdi,S. and Roth,M. 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