182-Glycoprotein I (apolipoprotein H) (anti-cardiolipin antibodies/lipid-protein complex/thrombosis)

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1 Proc. Natd. Acad. Sci. USA Vol. 87, pp , June 199 Medical Sciences Anti-phospholipid antibodies are directed against a coplex antigen that includes a lipid-binding inhibitor of coagulation: 182-Glycoprotein (apolipoprotein H) (anti-cardiolipin antibodieslipid-protein coplexthrobosis) H. PATRCK MCNEL*, RCHARD J. SMPSONt, COLN N. CHESTERMAN*, AND STEVEN A. KRLS*t *University of Ne South Wales, School of Medicine, Saint George Hospital, Kogarah, 2217 Australia; and Joint Protein Structure Laboratories, The Ludig nstitute for Cancer Research and The Walter and Elia Hall nstitute for Medical Research, Parkville, 35 Australia Counicated by K. Frank Austen, March 15, 199 ABSTRACT Anti-phospholipid (apl) antibodies that exhibit binding in cardiolipin (CL) ELSA can be purified to >95% purity by sequential phospholipid affinity and ionexchange chroatography. Hoever, these highly purified apl antibodies do not bind to the CL antigen hen assayed by a odified CL ELSA in hich the blocking' agent does not contain bovine seru, nor do they bind to phospholipid affinity coluns. Binding to the phospholipid antigen ill only occur if noral huan plaa, huan seru, or bovine seru is present, suggesting that the binding of apl antibodies to CL requires the presence of a plasaseru cofactor. Using sequential phospholipid affinity, gel-filtration, and ion-exchange chroatography, e have purified this cofactor to hoogeneity and shon that the binding of apl antibodies to CL requires the presence of this cofactor in a dose-dependent anner. N-te l region sequence analysis of the olecule has identified the cofactor as 32-glycoprotein (2GP) (apolipoprotein H), a plasa protein knon to bind to anionic phospholipids. These findings indicate that the presence of 2GP is an absolute requireent for antibody-phospholipid interaction, suggesting that bound J2GP fors the antigen to hich apl antibodies are directed. Recent evidence indicates that B2GP exerts ultiple inhibitory effects on the coagulation pathay and platelet aggregti. nterference ith the function of J2GP by apl antibodies could explain the throbotic diathesis seen in association ith these antibodies. Anti-phospholipid (apl) antibodies are autoantibodies that can be detected in plasa or seru in solid-phase iunoassays in hich negatively charged phospholipids, ost coonly cardiolipin (CL), are used as the antigen (1, 2). We have previously described a siple to-step procedure for purifying apl antibodies fro plasa (or seru) by sequential phospholipid affinity and cation-exchange chroatography, yielding specific iunoglobulin of >95% purity (3). These antibodies exhibit typical binding in CL ELSA but do not possess lupus anticoagulant (LA) activity in phospholipid-dependent clotting tests. Recently, e have also shon that plasa can be resolved by ion-exchange chroatography into fractions containing either anticardiolipin (acl) antibodies or antibodies ith LA activity, strongly suggesting that acl and LA antibodies represent distinct antibody subgroups (4). Although antibodies binding CL in iunoassays also generally bind all anionic phospholipids (2, 4), and hence are best referred to as apl antibodies, e hereafter refer to this group as acl antibodies in distinction to LA, hich ay also be apl antibodies but appear to be directed against a different antigen (4). The publication costs of this article ere defrayed in part by page charge payent. This article ust therefore be hereby arked "advertiseent" in accordance ith 18 U.S.C solely to indicate this fact. 412 We have previously noted that hen acl antibodycontaining fractions derived fro ion-exchange chroatography of plasa ere applied to phosphatidylserine or CL affinity coluns, there as no binding of the antibody despite the fact that hen plasa containing these antibodies as applied to these coluns, acl antibodies could be purified. This suggested that there as a cofactor also present in plasa or seru that as required for acl antibodies to bind to the affinity coluns. Addition of noral (acl antibody negative) plasa to the ion-exchange fractions resulted in acl binding to the coluns supporting this hypothesis (4). n this report, e have further investigated this phenoenon. We have found that the plasa cofactor is also required for acl antibodies to bind CL in a odified iunoassay in hich bovine seru (hich also contains the cofactor) is excluded. We have been able to purify the cofactor to hoogeneity and identify it as 2-glycoprotein (p32gp), also knon as apolipoprotein H. These results ay change our understanding of the biology of acl antibodies. MATERALS AND METHODS Plasa and Patients. Citrated platelet-free plasa as prepared by adding freshly dran blood fro venipuncture into tubes containing 11th final vol of.11 M trisodiu citrate, iediate centrifugation at 25 x g for 15 in, and filtration through a.22-pu Millipore Millex filter (4). acl antibodies ere purified fro to patients ith antiphospholipid syndroe hose plasa contained high levels of acl antibodies and strong LA activity. Clinical details and coplete coagulation screen on these to patients have been previously published (4). Plasas fro to other patients ith systeic lupus erytheatosus but ithout histories of throboses ere subjected to ion-exchange chroatography. The plasa cofactor as purified fro a noral healthy 31-year-old ale hose plasa as negative for acl antibodies and LA activity. on-exchange Chroatography of Patient Plasa. Patient plasa as extensively dialyed against.5 M acetate.5 M NaCl buffer, ph 4.8, centrifuged, then run on a Sepharose fast flo cation-exchange colun (Pharacia) using a Pharacia fast protein liquid chroatography (FPLC) syste ith a 15-l linear gradient of -1%c eluting buffer (.5 M acetate.65 M NaCl, ph 5.2) over 5 hr as described (4). Fractions ere assayed for acl antibodies by a standard CL ELSA (5) and acl antibody-positive fractions ere dialyed against.1 M phosphate-buffered.15 M saline (ph 7.2) (PBS) (Dulbecco A). Abbreviations: 32GP,,32-glycoprotein ; CL, cardiolipin; acl, anti-cl; apl, anti-phospholipid; LA, lupus anticoagulant. tto ho reprint requests should be addressed.

2 Medical Sciences: McNeil et al. CL Affinity Chroatography. A odification of the previously described ethod (3) for phosphatidylserine affinity chroatography as used. CL (5.6 g in ethanol) (Siga) as placed in a glass scintillation vial and the solvent as evaporated under a strea of nitrogen. Cholesterol (2.32 g) (BDH) and dicetyl phosphate (.44 g) (Siga) ere added and the lipids ere redissolved in chlorofor. The solvent as again evaporated, 5,ul of ethanol as added, and the vial as capped and iersed in boiling ater and sirled until the lipids ere dissolved. Five illiliters of a 15% acrylaide5% bisacrylaide (Bio-Rad) solution as added and the lipidacrylaide ixture as vigorously ixed in a Vortex, then rapidly polyeried by adding 1 tul of aoniu persulfate (14 gl), 2.5,1 of TEMED (both fro Bio-Rad), and then left at roo teperature overnight. The rigid gel as hoogenied by using a hand-operated loosefitting Teflon pestle and loaded into an epty Pharacia FPLC HR 11 glass colun and equilibrated ith.1 M phosphate.5 M NaCl, ph 7.2. Plasa as diluted 1:5 final volue ith this buffer and infused through the colun at.5 lin, after hich the colun as ashed at.8 lin until the absorbance at 28 n of the fall-through as <.1 unit. A 4-l linear gradient fro % to 1% of eluting buffer (.1 M phosphate1. M NaCl, ph 7.2) over 5 in as applied and eluted fractions containing protein ere collected. on-exchange fractions ere applied undiluted and chroatographed as described above. Standard acl Antibody unoassay. The ELSA for acl antibodies previously described (2) ith inor odifications (5) as used. When using polyvalent second antibody, serial dilutions of a knon positive plasa ere included on each plate. The acl antibody level of the test saples as expressed in acl antibody units ith 1 units being the absorbance at 45 n of the positive saple at 1:5 dilution. Modified acl Antibody unoassay. The above standard CL ELSA as odified as follos. Milk poder (1%) (Diploa, Unigate Australia, Dandenong, Australia).3% gelatin (Ajax Cheicals, Ne South Wales, Australia)PBS as used for the blocking step, saples ere added diluted in.3% gelatinpbs, and the second antibody as diluted in 1% bovine seru albuinpbs instead of 1% adult bovine serupbs, hich as used in each of the above steps in the standard assay. All other details reained identical to the standard CL ELSA described above. Affinity Purification of acl Antibodies. Plasa fro patients ith high levels of acl antibodies as chroatographed on the CL affinity colun as described above. The protein eluted fro this colun as dialyed overnight against.5 M acetate.5 M NaCl, ph 4.8, then applied to a Mono-S cation-exchange colun (Pharacia) using a FPLC syste. A 15-l linear gradient fro % to 1% of eluting buffer.5 M acetate.65 M NaCl, ph 5.2, over 3 in as applied and highly purified acl antibodies eluted at 3% eluting buffer (3). Purification of the Plasa Cofactor. Noral plasa as chroatographed on the CL affinity colun as described above. Fractions containing the eluted protein ere concentrated in an Aicon concentrator ith a YM 1 ebrane and 2 kpa pressure, then dialyed against.1 M phosphate1 M NaCl, ph 7.2, and further concentrated to 2,ul in a Micro-Pro-DiCon dialying concentrator (Bio-Molecular Dynaics, Beaverton, OR) ith a PA-1 ebrane. The concentrated CL colun eluant as applied to a Pharacia Superose gel-filtration colun operating ith a FPLC syste at.4 lin in a buffer of.1 M phosphatei M NaCl, ph 7.2. Fractions fro this colun that ere found to have cofactor activity (as described in Results) ere pooled and dialyed overnight against.5 M acetate.5 M NaCl, ph 4.8, then applied to a Pharacia Mono-S cationexchange colun and eluted ith.5 M acetate.65 M Proc. Natl. Acad. Sci. USA 87 (199) 4121 NaCl as described above for the purification of acl antibodies. Fractions found to have cofactor activity ere pooled and dialyed against PBS and then stored in aliquots at -7C. Aino Acid Sequencing. Autoated Edan degradation of purified cofactor as perfored ith an Applied Biosystes sequencer (odel 477A) equipped ith an on-line phenylthiohydantoin aino acid analyer (odel 12A). Total aino acid derivatives fro the sequencer ere injected onto the liquid chroatograph by using a odified saple transfer device described elsehere (6). Polybrene as used as a carrier (7). Heparin Affinity Chroatography. acl antibody-positive plasa, or affinity-purified acl antibody, andor purified cofactor ere applied to a colun packed ith 4 l of heparin-sepharose CL-4B (Pharacia) in.1 M phosphate.5 M NaCl buffer, ph 7.2, and bound protein as eluted ith.1 M phosphatei M NaCl, ph 7.2. Other Methods. Saples for SDSPAGE ere run on a 5-15% linear gradient gel ith a 3% stacking gel (8). After electrophoresis, the gel as stained ith Cooassie blue. Protein deterination as by the ethod of Lory et al. (9). Vitain K-dependent coagulation factor-depleted plasa as prepared by ixing.1 l of 5% aluinu hydroxide suspension (BDH) ith 1 l of plasa and inverting the tube every inute for 15 in before centrifuging the precipitate. RESULTS CL Affinity Chroatography of acl Antibody-Containing on-exchange Fractions fro Plasa. As previously described (4), plasa containing acl antibodies and LA activity can be separated into subgroups by cation-exchange chroatography. When acl antibody-containing fractions ere infused through the CL affinity colun, there as no binding ith the colun fall-through containing equivalent aounts of acl antibody as the applied fraction. When noral plasa as added to the ion-exchange fractions in a ratio of 1:1 (volvol) and the ixture as infused through the CL affinity colun, -3% of the applied acl antibodies absorbed to and could be eluted fro the colun (Table 1). Noral plasa that had been ixed ith aluinu hydroxide to reove vitain K-dependent coagulation factors also produced a siilar effect. Finally, a fraction derived fro noral plasa as purified (see belo) and addition of this "cofactor" fraction (protein concentration, 2,tgl) in a ratio of 1:5 (volvol) resulted in the absorption of 74% of the applied acl antibodies. Binding of acl Antibody-Containing on-exchange Fractions and Affinity-Purified acl Antibodies in a Modified acl Antibody unoassay. The above results suggested a plasa dependency for the binding of acl antibodies to CL in the affinity colun. Hoever, these acl antibody- Table 1. Chroatography of acl antibody-containing ion-exchange fractions on CL affinity colun Aount of acl antibody Total applied, Fall-through, Saple units units (%) E (96) E + noral P1* 12 8 (66) TE + adsorb P1* 12 9 (75) E + 832GPt (26) E, ion-exchange fraction; P1, plasa; adsorb, Al(OH)3 adsorbed. % of total applied acl antibody recovered in fall-through is given in parentheses. *One illiliter of plasa as added to 1 l of E. tfour hundred icrogras of f32gp (equivalent to 2 l of plasa) as added to 1 l of E.

3 4122 Medical Sciences: McNeil et al. containing fractions and affinity-purified acl antibodies exhibit typical binding in standard solid-phase CL iunoassays in the absence of plasa (3). These assays use bovine seru as the blocking agent and diluent, hich could conceivably also contain the plasa factor necessary for antibody binding to CL. To test this, e replaced the bovine seru ith gelatin as the diluent and ilk podergelatin as the blocking agent. When acl antibody-containing ionexchange fractions or affinity-purified acl antibodies ere tested in this odified CL iunoassay, there as no binding at all. At 1:5 dilution, acl antibody-positive plasa exhibited identical binding as in the standard CL ELSA and noral plasa as negative. Binding of antibodies in the ion-exchange fractions or affinity-purified preparations did occur if noral plasa as added at 1:5 dilution. An identical effect as also found if bovine seru as added to these fractions (Fig. 1). Thus, purified acl fractions ill bind in the standard CL ELSA since the cofactor is provided by bovine seru. This odified CL ELSA provided a convenient ethod to detect cofactor activity in fractions derived fro noral plasa by testing saples containing a ixture of affinitypurified acl antibodies and the fraction containing an unknon aount of cofactor. n the absence of cofactor, the affinity-purified acl antibodies ould not bind and any binding as evidence of cofactor activity in the test fraction. As described belo, e purified the cofactor activity fro noral plasa, and the addition of this fraction to affinitypurified acl antibodies or acl antibody-containing ionexchange fractions resulted in binding of these antibodies in the odified CL ELSA (Fig. 1). Purification and dentifcation of the Cofactor in Noral Plasa. Noral plasa as chroatographed on the CL affinity colun and the fractions ere tested for cofactor activity as described above by the odified CL ELSA. Cofactor activity as found in the eluted protein, indicating that the factor bound to anionic phospholipid even in the absence of acl antibodies. These fractions ere concentrated and chroatographed on a Superose 12 gel-filtration colun in 1 M NaCl buffer. Cofactor activity as found in a peak eluting at a Ka, of.35, hich corresponded to an apparent olecular ass of 67 kda on this colun (Fig. 2). The fractions containing this peak ere pooled, dialyed 2 CJ cr C', 3 3 an 1 J STD CL ELSA 2 MOD CL ELSA P.PL N.PL E.F -J EL + LL U < AP. AB FG. 1. Binding of various saples in standard and odified CL ELSA. E.F, acl antibody-containing ion-exchange fractions; AP.AB, affinity-purified acl antibodies; N.PL, noral plasa; ABS, adult bovine seru; COF, purified cofactor; P.PL, patient plasa. ABS and COF are negative hen tested alone (data not shon). E C cu C.) a: co tn FRACTON NUMBER FG. 2. Gel filtration on a Pharacia Superose 12 HR 13 colun of the eluant fro CL affinity colun chroatography of noral plasa. Buffer as.1 M phosphatei M NaCi. Cofactor activity as detected in the large peak indicated by the boldface arro. against the ion-exchange starting buffer, and chroatographed on a Mono-S colun. Cofactor activity as found in a late peak eluting at.5 M NaCl (Fig. 3). The final preparation fro the ion-exchange colun as found to be highly purified ith a single band on SDSPAGE of apparent olecular ass 5 kda under both reduced and nonreduced conditions (Fig. 4). This band corresponded to a band previously found in the CL or phosphatidylserine affinity colun eluant hen acl antibody-containing plasa as chroatographed on those coluns (also shon in Fig. 4). The N-terinal aino acid sequence of the cofactor is shon in Fig. 5. A coputer search of current data base releases shos this sequence is identical to the N-terinal sequence of the plasa protein P2GP, also knon as apolipoprotein H. There ere no other proteins that exhibited hoology to this sequence except a plasa protein purified by Canfield and Kisiel (1), tered activated protein C.8 T c.6 co cu W 4. X. 2 Proc. Natl. Acad. Sci. USA 87 (199) o 4, FRACTON NUMBER 1 cr LL LL 3 5 c FG. 3. Cation exchange on a Pharacia Mono-S HR 55 colun of the cofactor-containing fractions fro Fig. 2. Starting buffer as.5 M acetate.5 M NaCl, ph 4.8. Eluting buffer as.5 M acetate.65 M NaCl, ph 5.2. Dashed line, eluting buffer gradient. Cofactor activity as detected in the late peak indicated by the arro. - -J

4 Medical Sciences: McNeil et al. Proc. Natl. Acad. Sci. USA 87 (199) 4123 kda 2_ b '*. o C) o M. 24.8' _ 97_. 66_ FG. 4. SDSPAGE on a 5-15% linear gradient gel under nonreducing conditions stained ith Cooassie blue. Lane 1, eluant fro CL affinity colun chroatography of acl antibody-positive plasa containing a broad band of gg acl (15 kda) and cofactor (5 kda). Lane 2, purified cofactor obtained fro noral plasa folloing sequential CL affinity, gel filtration (Fig. 2), and cationexchange chroatography (Fig. 3). binding protein, hich has subsequently been reported to be identical to 32GP (11, 12). nteraction Beteen Purified f2gp and Affinity-Purifiled acl Antibodies in the Modified CL ELSA. Since affinitypurified acl antibodies do not bind CL in the odified CL ELSA unless 182GP is present (Fig. 1), e exained the binding of sequential dilutions ofthe antibody in the presence of sequential dilutions of glycoprotein purified as described above. These results are shon in Fig. 6a. n the presence of P2GP (8 pgl), serial dilutions of acl antibody sho a binding curve typical of that found ith the standard CL ELSA. With increasing dilutions of 182GP, binding decreases draatically at all dilutions of antibody ith virtually no binding hen the glycoprotein is reduced to 1 pgnl. The dashed line in Fig. 6a accounts for serial dilution of both factors. Dilution ofacl antibody-positive plasa is shon in Fig. 6b. The solid line shos the failiar binding curve in a standard CL ELSA in hich the presence of adult bovine seru ensures adequate cofactor at all antibody dilutions. The dashed line shos the binding curve hen acl antibodypositive plasa is diluted in the odified CL ELSA. There is virtually no binding of antibody once plasa is diluted to 1:2, corresponding to a P2GP level of =1 pugl. Heparin Affinity Chroatography. Affinity-purified acl antibodies did not bind in a heparin-sepharose affinity colun ith -6% of the applied acl antibody recovered in the fall-through fractions, but no antibody as eluted ith 1 M NaCl. Purified,82GP bound to the heparin-sepharose hen applied to the colun. When a ixture of affinity-purified acl antibodies and purified 132GP as infused through the heparin-sepharose colun, the fall-through contained acl but not,82gp and the eluted protein contained f2gp but not acl. When plasa containing acl antibodies as infused NH 2-Gly-Arg-Thr-X-Pro-Lys-Pro-Asp-Asp-Leu-Pro-Phe Ser-Thr-Va l-va l-pro-leu-x-thr-phe-tyr-glu-pro- FG. 5. N-terinal aino acid sequence of the purified cofactor. X, residues cannot be identified unabiguously... 2 C a i 24 ca..a a:.4- cn PLASMA D LUT ON A C AN*N TBD (ug. A. P. acl ANTBODY tugl).3 FG. 6. (a) Binding in the odified CL ELSA of serial dilutions of affinity-purified (A.P.) acl antibodies in the presence of serial dilutions of cofactor. Dashed line, extrapolated binding curve based on dilution of both antibody and cofactor. (b) Binding in the standard CL ELSA (solid line) and odified CL ELSA (dashed line) of serial dilutions of acl antibody-positive plasa. through heparin-sepharose, the fall-through fractions contained equivalent quantities of acl antibody to the applied plasa, but the eluted protein contained,82gp and not acl antibody. DSCUSSON We have clearly deonstrated in to systes that a plasa cofactor is required for acl antibodies to bind CL. We have purified this cofactor and identified it as a knon plasa protein, (32GP. n the affinity chroatography syste, acl antibodies contained in fractions derived fro ion-exchange of plasa did not bind to iobilied CL despite doing so hen present in plasa, but binding did occur if noral plasa as added to the ion-exchange fraction. This as the first suggestion that a plasa cofactor as involved in acl antibody-cl interactions, and e have confired this by isolating this cofactor activity to a single plasa protein, f32gp (Table 1). Having deonstrated this requireent, hy these ionexchange fractions bound CL in the standard CL ELSA as probleatic. One explanation as that the cofactor or a siilar olecule as also present in the bovine seru diluent used in the iunoassay. We confired this by odifying the CL ELSA, oitting bovine seru and using gelatin as the diluent. n this assay, acl antibody-containing ionexchange fractions and affinity-purified acl antibodies did not bind CL unless noral plasa or bovine seru as added (Fig. 1). Once again, in this syste, the cofactor activity as isolated to f32gp. This as a consistent finding in 11 separate acl antibody-containing ion-exchange fractions fro four patients consecutively studied, and three affinity-purified acl antibody preparations [to gg and one gm] (data not shon). The odified CL ELSA proved to be a convenient syste to exaine the cofactor effect in ore detail. We found the action of f82gp on acl antibody binding to CL as dose dependent ith a steep dilution effect, such that there as virtually no antibody binding at any dilution hen j32gp as

5 4124 Medical Sciences: McNeil et al. present at 1 pugl or less (Fig. 6a). Since the noral plasa level of 82GP is 2 Agl, this figure corresponded exactly to the results of diluting acl antibody-positive plasa in the odified CL ELSA, here there as a rapid drop-off in binding, hich becae negative at 1:2 dilution, or at a 32GP level of 1 ugl, despite the presence of appreciable aounts of acl antibody (Fig. 6b). When plasa containing gg acl antibodies is chroatographed on CL or PS affinity coluns, to ajor protein bands are eluted (3). One of these is gg, and the other is 132GP (Fig. 4). Since e purified,82gp fro noral plasa by using the CL affinity colun, it is clear that f2gp binds to anionic phospholipid coluns in both the presence and absence of acl antibodies. Yet these antibodies do not bind CL in the absence of p2gp. Purified P2GP but not acl bound to a heparin-sepharose colun, but acl antibodies did not bind to this colun even in the presence of bound 132GP. Thus, acl antibodies recognied P2GP bound to anionic phospholipid, but not f32gp bound to heparin, indicating that both phospholipid and glycoprotein coprise the epitope to hich these antibodies are directed. These results suggest that acl antibodies are directed against either a coplex consisting of f32gp bound to anionic phospholipid, or a cryptic epitope fored during the interaction of 82GP ith phospholipid, but not,82gp independent of the presence of phospholipid. (32GP is a plasa,82-globulin first described in 1961 (13). The coplete aino acid sequence as deterined in 1984 (11). t is coposed of 326 aino acids and has a unique sequence ith abundant proline residues, ultiple disulfide bridges, and a high carbohydrate content (19%) resulting in an apparent olecular ass of 5 kda (11). The physiologic function of this glycoprotein is uncertain, but it is knon to bind to lipoproteins (14), anionic phospholipids (15), platelets (16), heparin (17), DNA (18), and itochondria (19). The concentration of 132GP in plasa is -2,ugl and 4%o is associated ith lipoproteins of various classes (14). Because of this, 2GP has been designated apolipoprotein H (2). n recent years, P2GP has been found to inhibit the intrinsic coagulation pathay (12, 21) and to bind to platelets (16) and inhibit ADP-ediated platelet aggregation (22). Schousboe (12) has suggested that one function of 132GP is to bind to and neutralie negatively charged acroolecules that ight enter the bloodstrea and thus diinish unanted activation of blood coagulation. The finding that acl antibodies are directed against an antigen that includes f32gp opens ore avenues to our understanding of these autoantibodies. t provides an explanation for acl antibodies binding equally ell to all anionic phospholipids despite various structures (2, 4). n addition, since 132GP appears to inhibit the intrinsic pathay of coagulation and ADP-dependent platelet aggregation, these Proc. Natl. Acad. Sci. USA 87 (199) findings raise the possibility that acl antibodies interfere ith f2gp function in vivo, thereby predisposing to a prothrobotic diathesis. Furtherore, if(32gp1 binds anionic acroolecules, this could include those derived fro infectious organiss. Foreign antigen coplexed ith p2gp could be the iunogenic stiulus for the production ofacl antibodies, these being a ell-recognied occurrence in a nuber of infectious diseases. Further studies directed at studying acl antibody-182gp interactions ay add to our understanding of these autoantibodies. H. P. McNeil is the recipient of a Postgraduate Medical Research Scholarship fro the National Health and Medical Research Council of Australia. This ork as supported in part by grants fro the National Health and Medical Research Council of Australia. 1. Harris, E. N., Gharavi, A. E., Boey, M. L., Patel, B. M., Mackorth-Young, C. G., Loiou, S. & Hughes, G. R. V. (1983) Lancet ii, Gharavi, A. E., Harris, E. N., Asherson, R. A. & Hughes, G. R. V. (1987) Ann. Rheu. Dis. 46, McNeil, H. P., Krilis, S. A. & Chesteran, C. N. (1988) Throb. Res. 52, McNeil, H. P., Chesteran, C. N. & Krilis, S. A. (1989) Br. J. Haeatol. 73, McNeil, H. P., Chesteran, C. N. & Krilis, S. A. (1988) Throb. Res. 52, Begg, G. S. & Sipson, R. J. (1989) in Techniques in Protein Cheistry, ed. Hugh, T. E. (Acadeic, San Diego), pp Klapper, D. G., Wilde, C. E. & Capra, J. D. (1978) Anal. Bioche. 85, Laeli, U. K. (197) Nature (London) 227, Lory, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Che. 193, Canfield, W. M. & Kisiel, W. (1982) J. Clin. nvest. 7, Loier, J., Takahashi, N. & Putna, F. W. (1984) Proc. Natl. Acad. Sci. USA 81, Schousboe,. (1985) Blood 66, Schulte, H. E., Heide, H. & Haupt, H. (1961) Naturissenschaften 48, Pol, E. & Kostner, G. M. (1979) FEBS Lett. 12, Wur, H. (1984) nt. J. Bioche. 16, Schousboe,. (198) Throb. Res. 19, Pol, E., Wur, H. & Kostner, G. M. (198) nt. J. Bioche. 11, Kroll, J., Larsen, J. K., Loft, H., Eban, M., Wallevik, K. & Faber, M. (1976) Bioche. Biophys. Acta 434, Schousboe,. (1979) Bioche. Biophys. Acta 579, Nakaya, Y., Schaefer, E. J. & Breer, H. B. (198) Bioche. Biophys. Res. Coun. 95, Nipf, J., Bevers, E. M., Boans, P. H. H., Till, U., Wur, H., Kostner, G. M. & Zaal, R. F. A. (1986) Bioche. Biophys. Acta 884, Nipf, J., Wur, H. & Kostner, G. M. (1987) Atherosclerosis 63,

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