Proc. Nati. Acad. Sci. USAVol. 89, pp. 10193-10197, November 1992Medical Sciences

Homocysteine and other sulfhydryl compounds enhance the bindingof lipoprotein(a) to fibrin: A potential biochemical link betweenthrombosis, atherogenesis, and sulflhydryl compound metabolism

(atherosderosis/vascular disease/ m /hyperhonocysteinemia)

PETER C. HARPEL, VICTOR T. CHANG, AND WOLFGANG BORTHDepartment of Medicine, Division of Hematology, Mount Sinai Medical Center, One Gustave Levy Place, New York, NY 10029; and the Specialized Centerfor Research in Thrombosis, Cornell University Medical College, New York, NY 10021

Communicated by Earl W. Davie, July 23, 1992

ABSTRACT We have previously shown that lipoprotein(a)[Lp(a)], an atherogenic lipoprotein that contains apolipopro-tein(a), which shares partial structural homology to plasmin-ogen, binds to a plasmin-modifled fibrin surface, and we havepostulated that this interaction may be atherogenic. Moderateelevations in blood homocysteine, a relatively common condi-tion, predispose to premature atherosclerosis. The reasons forthis are not established. We now report that homocysteine, atconcentrations as low as 8 jM, sigcantly ias theaMnity of Lp(a) for fibrin. Homocysteine induces a 20-foldincrease in the affinity between Lp(a) and plasmin-treatedfibrin and a 4-fold increase with unm ed fibrin. Lp(a)binding Is inhibited by e-am aproic acid, i n lysinebinding site specificity. Homocysteine does not enh thebndiny of Lp(a) to other surface-bound proteins. Cysteine,glutathione, and N-acetylcysteine also increase the affinitybetween Lp(a) and fibrin. Homocysteine does not affect thebinding oflow density lipoprotein or pasminogen to fibrin, nordoes it alter the gel-filtration elation pattern of Lp(a). Immu-noblot analysis documents the fact that homocysteine partiallyreduces Lp(a). These results suggest that homocysteine altersthe intact Lp(a) particle so as to increase the reactivity of theplasminogen-like apolipoprotein(a) portion of the molecule.The observation that sulthydryl amino acids increase Lp(a)binding to fibrin suggests a biochemical relationship betweensulfhydryl compound metabolism, thrombosis, and atherogen-esis.

Lipoprotein(a) [Lp(a)], identified by Berg in 1963 (1), consistsof a low density lipoprotein (LDL) particle enveloped by aunique apolipoprotein, apolipoprotein(a) [apo(a)], disulfidelinked to the apolipoprotein B-100 (apoB-100) moiety ofLDL(2). Apo(a) shows remarkable homology with plasminogen (3,4), and considerable size heterogeneity has been reported dueto allelic differences in the number of its tandemly repeatedsequences that resemble kringle four of plasminogen (5).Approximately 20%o of the population have elevated Lp(a)plasma levels (6). Epidemiologic studies have shown thatincreased plasma levels of Lp(a) represent a risk factor forcoronary heart disease (7-9), occlusion of saphenous veinbypass grafts (10, 11), and stroke (8, 12, 13). Because of itshomology with plasminogen, it has been proposed that Lp(a)may compete for binding with fibrin (14-17) and cell-surfaceplasminogen (15, 18, 19), thereby creating a procoagulant andthrombogenic milieu.

Lp(a) is demonstrable in atheromatous plaques (20-22) andimmunohistochemical studies by Wolf et al. show that Lp(a)colocalizes extracellularly with fibrin in these lesions (23).Plasmin digestion ofhuman plaques extracts Lp(a) and fibrin

degradation products, suggesting that Lp(a) is physicallyassociated with fibrin in the arterial wall (24). A furtherrationale for mechanisms that may link thrombosis to ath-erogenesis has been provided by our studies showing thatLp(a) displays strong affinity for plasmin-modified fibrinsurfaces in a lysine binding site-dependent fashion (14). Thebinding of Lp(a) to fibrin thus provides a mechanism forincorporating Lp(a) into the blood vessel wall and the ather-omatous plaque. Based on our observations that Lp(a) bindsto plasmin-modified fibrin we have initiated studies to iden-tify agents that can alter the Lp(a)-fibrin reaction. We nowreport that homocysteine as well as other sulfhydryl com-pounds enhance the binding of Lp(a) to surface-immobilized,plasmin-treated fibrin.*

MATERIALS AND METHODSReagents. DL-homocysteine and L-cysteine were from

ICN. S-Adenosyl-L-homocysteine, L-methionine, L-homo-cystine, glutathione, and dithiothreitol were from Sigma.

Lipoprotein and Protein Purification. Lp(a) and LDL werepurified from fresh plasma as described (14) and the concen-tration was expressed as protein as determined by the Lowryprocedure. Lp(a) lacking apo(a) was produced by heparinaffinity chromatography as described (26). Lp(a) was bio-tinylated using NHS-LC-biotin (Pierce). Fibrinogen (Kabitype L) was depleted of fibronectin and plasminogen asdetailed (27). Glu-plasminogen and plasmin were obtainedfrom American Diagnostica (Greenwich, CT). a2-Plasmininhibitor and a2-macroglobulin were purified as detailed (28).Fibronectin and platelet thrombospondin were provided byDominick Falcone and Roy Silverstein (Cornell UniversityMedical College, New York) and recombinant apo(a) was agenerous gift from Dan Eaton (Genentech).

Antisera. Rabbit anti-Lp(a) IgG was labeled with alkalinephosphatase as detailed (14, 28). At the dilutions used for theLp(a) ELISA detailed below, the labeled antisera detectedLp(a) and apo(a) but not LDL.Binding of Lp(a) to Immobilized Proteins by ELISA. Inter-

action of Lp(a) with immobilized fibrin was studied asdescribed (14). Lp(a) was preincubated with homocysteine inTris/Tween buffer alone or containing 50 mM E-aminoca-proic acid (eACA) and then added to the ELISA plate wellscoated with fibrinogen that had been treated with thrombinand plasmin. Similar experiments were done with varioussulfhydryl or sulfur-containing compounds. Rabbit anti-Lp(a) IgG labeled with alkaline phosphatase was then added.After an 18-hr incubation, the substrate, p-nitrophenyl phos-

Abbreviations: Lp(a), lipoprotein(a); LDL, low density lipoprotein;apo(a), apolipoprotein(a); EACA, e-aminocaproic acid.*This study was presented in part at the American Society ofHematology 32nd Annual Meeting, December 1990, and has ap-peared in abstract form (25).

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

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phate (Sigma) was added. Color development at 405 nm was

followed in a microplate reader (Molecular Devices, PaloAlto, CA). The results were expressed as OD units x 10-3 per

min using software supplied by the manufacturer.To convert OD units to Lp(a) bound to the fibrin surface,

recombinant apo(a) was radiolabeled with 125I by the McFar-lane technique (29). The binding of labeled recombinantapo(a) to plasmin-modified fibrin was quantified by directdetermination of the amount of radiolabeled recombinantapo(a) bound in conjunction with ELISA. The number ofODX 10-3 units per min plotted against pmol of recombinantapo(a) bound was linear to 160 units; y = 426855.6fx + 1.8954(r = 0.99). Utilizing these data, Lp(a) binding was analyzedby the EBDA ligand radioligand binding programs of G. A.McPherson (Elsevier-Biosoft, Cambridge, U.K.). Nonspe-cific binding was considered to be the binding of Lp(a) in thepresence of 50 mM EACA.The binding of Glu-plasminogen to plasmin-treated fibrin

was examined by preincubating plasminogen with homocys-teine for 2 hr before addition to the fibrin surface. Binding ofplasminogen was detected by using rabbit anti-plasminogenIgG labeled with alkaline phosphatase (28). ELISA platewells were also coated with albumin, fibronectin, plateletthrombospondin, and a2-macroglobulin.

Gel-Filtration Chromatography and Immunoblot Analysisof Homocysteine-Treated Lp(a). Lp(a) incubated with homo-cysteine or buffer was analyzed by Sephacryl S400 high-resolution gel column chromatography in 0.05 M TrisHClbuffer (pH 8.0) containing 0.1 M NaCl, 1 mM EDTA, and0.02% sodium azide. Separation was performed at a flow of0.3 ml/min. Absorbance readings at 280 nm were convertedto concentrations of Lp(a) using an extinction coefficient of17.5 as derived from the Lowry assay. Fractions were testedfor binding to plasmin-treated fibrin by ELISA as detailedabove. The OD x 10-3 units were converted to ,ug of Lp(a)by using a Lp(a) binding curve constructed by adding serialdilutions of Lp(a) (0-16 ,ug/ml) to the immobilized fibrin.Immunoblot analysis of Lp(a) was performed by minormodifications of methods detailed (30).

RESULTSEffect of Homocysteine on Binding of Lp(a) to Immobilized

Fibrin. Homocysteine was found to substantially increase theaffinity of Lp(a) for both the plasmin-modified fibrin as wellas for the untreated fibrin surface (Fig. 1). The largest effectof homocysteine was on the binding of Lp(a) to plasmin-modified fibrin (Fig. 1 Left). The dissociation constant (Kd)for the interaction between Lp(a) and plasmin-treated fibrinwas 0.18 nM in the presence of homocysteine and 3.67 nM inits absence (Table 1). Homocysteine also enhanced thebinding of Lp(a) to fibrin that was not degraded by plasmin(Fig. 1 Right). With homocysteine, the Kd for interactionbetween Lp(a) and fibrin was 4.15 nM, similar to the disso-ciation constant for the interaction of Lp(a) with plasmin-modified fibrin. The lowest affinity (Kd = 15.3 nM) wasobserved for undegraded fibrin in the absence of homocys-teine. Plasmin treatment of the fibrin surface increased thenumber of Lp(a) binding sites =5-fold. In each instance, thepresence of EACA inhibited the binding of Lp(a) or ofhomocysteine-treated Lp(a) to plasmin-treated fibrin.Lp(a-), a Lp(a) particle depleted of apo(a), or LDL from thesame donor showed poor affinity for fibrin as well as forplasmin-modified fibrin, and the binding remained unaffectedby homocysteine (data not shown). At saturating concentra-tions of Lp(a), approximately one Lp(a) particle was boundto 100 molecules of fibrin monomer. The ability of homocys-teine or EACA to reverse the binding ofhomocysteine-treatedLp(a) was also tested. After Lp(a) had been incubated withthe plasmin-modified fibrin surface for 90 min, buffer con-

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FIG. 1. Effect of homocysteine on binding of Lp(a) to immobi-lized fibrin. (Left) Microtiter plate wells were coated with fibrinogenand subsequently treated with thrombin and plasmin (Plasmin +).Increasing concentrations of Lp(a) preincubated 2 hr with either 2mM homocysteine (0), buffer (o), homocysteine with 50 mM eACA(v), or buffer with EACA (A) was added. After incubation for 90 min,the wells were washed, and the binding of Lp(a) was determined byELISA. (Right) Lp(a) treated as described above was added tountreated (Plasmin -) fibrin-coated wells, and binding was measuredas described above.

taining 2 mM homocysteine, homocystine, or 0.05 M eACAwas added for an additional 30 min. No decrease in theamount of Lp(a) bound was detected, indicating that thesecompounds did not reverse Lp(a) binding. The failure ofeACA to dissociate Lp(a) from the fibrin surface suggests thatnonlysine binding site-dependent interactions occur betweenLp(a) and fibrin after complex formation.

Caution was applied in interpreting these observationssince it remained uncertain whether homocysteine treatmentproduced an increase in the number of exposed epitopes per

Lp(a) particle bound or whether the total number of Lp(a)particles deposited on the fibrin surface had increased. Theuse of an Lp(a)-biotin complex and an avidin-aikaline phos-phatase detection system showed a similar increase in Lp(a)binding in the presence of homocysteine and allowed us toconclude that homocysteine mediated an increase in theconcentration of Lp(a) on the fibrin surface.The effect of homocysteine on increasing Lp(a) binding to

fibrin was proportional to the concentration of the sulfhydrylcompound added (Fig. 2). The lowest concentration of ho-mocysteine examined, 8 juM, also increased Lp(a) binding incomparison to the buffer control (Fig. 2 Inset). In severalexperiments, we established that the homocysteine effectwas on the Lp(a) particle and not on the fibrin surface (datanot shown). Pretreatment of plasmin-degraded fibrin with

Table 1. Dissociation constants for Lp(a)-fibrin interactionPlasmintreatment Homocysteine Relativeof fibrin treatment Kd affinityNo No 1.53 x 10-8 M 1No Yes 4.15 x 10-9 M 3.7Yes No 3.67 x 10-9 M 4.2Yes Yes 1.83 x 10-10 M 83.6Fibrin adsorbed on microtiter plate wells was incubated with either

plasmin or buffer before addition of Lp(a). Lp(a) was preincubatedin 2 mM homocysteine or buffer before addition to the immobilizedfibrin. Dissociation constants (Kd) were calculated by the bindingprograms of G. A. McPherson (Elsevier-Biosoft). Relative affinityof the various interactions between Lp(a) and fibrin was calculatedrelative to the Kd for the interaction between fibrin and Lp(a).

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FIG. 2. Effect ofhomocysteine concentration on binding of Lp(a)to plasmin-modified fibrin. Lp(a) (0.6 nM) was preincubated 2 hr atroom temperature with serial dilutions of homocysteine as indicatedon the abscissa. These mixtures were applied to microtiter plate wellscoated with plasmin-modified fibrin for an additional 90 min, and theLp(a) bound was determined by ELISA. (Inset) Effect of 8 uMhomocysteine (HC) on binding of Lp(a) to plasmin-modified fibrin.Increasing concentrations of Lp(a) in buffer (abscissa) containing 8,uM homocysteine (m) or no homocysteine (A) was added to plasmin-modified fibrin. Lp(a) binding was determined by ELISA with a3-fold concentrated antibody probe to increase sensitivity; results areexpressed as OD x 10-3 per min (ordinate).

homocysteine had no enhancing effect on subsequent bindingof Lp(a). The addition of homocysteine to Lp(a) that wasalready bound to the plasmin-treated fibrin surface did notchange the signal produced by the alkaline phosphatase-labeled anti-Lp(a). The possibility that a sulfhydryl-disulfideexchange reaction between Lp(a) and a fibrin surface mightparticipate in the Lp(a)-fibrin binding reaction was ruled outby the demonstration that reduction and S-cysteinyl car-boxymethylation ofthe plasmin-treated fibrin surface prior tothe addition of Lp(a) did not alter the increase in bindingproduced by homocysteine.

Studies of the Specificity of the Homocysteine-Fibrin Inter-action. To study the specificity of the Lp(a)-fibrin interac-tion, Lp(a) with homocysteine or with buffer was added tomicrotiter plates coated with plasmin-modified fibrin, albu-min, fibronectin, platelet thrombospondin, or a2-macroglob-ulin (Fig. 3 Left). Only the plasmin-treated fibrin surfaceshowed an increase in Lp(a) binding in the presence ofhomocysteine. We also studied whether homocysteine mightaffect the binding of Glu-plasminogen. Plasmin treatment ofthe fibrin surface increased plasminogen binding in compar-ison to the nontreated fibrin (Fig. 3 Right). In contrast to theeffect shown with Lp(a), preincubation with homocysteinedid not increase the binding ofplasminogen to either plasmin-treated or untreated fibrin.Other sulfhydryl-containing compounds were also found to

increase the binding ofLp(a) to the plasmin-modified surface.Dithiothreitol (1 mM) was more effective in modulating Lp(a)binding than was homocysteine (2 mM) (Fig. 4). Cysteine,N-acetylcysteine, and glutathione also increased Lp(a) bind-ing, although the latter two appeared to be somewhat lesseffective. In contrast, sulfur-containing components of thetranssulfuration pathway (31) that lack the free sulfhydrylgroup including methionine, S-adenosylhomocysteine, andL-homocystine had no effect on Lp(a) binding. Oxidizeddithiothreitol was also ineffective.

Gel-Filtration Chromatography and Immunoblot Analysisof Homocysteine-Treated Lp(a). The nature of the homocys-teine-treated Lp(a) particle that bound to the fibrin surfacewas also examined by gel-filtration chromatography. There

0 t * -

0.0 0.2 0.4 0.6 0.8 1.0 0 35 70 105 140 175Lp(a), nM Plg, nM

FIG. 3. (Left) Binding of homocysteine-treated Lp(a) to variousimmobilized proteins. Microtiter plate wells were coated with fibrin-ogen that was treated with thrombin and plasmin as detailed in Fig.1. Alternatively, wells were coated with human albumin, fibronectin,platelet thrombospondin, or a2-macroglobulin. Lp(a) was subse-quently added in 2 mM homocysteine or in buffer, and binding wasdetermined by ELISA after a 90-min incubation. The Lp(a) bound inbuffer was subtracted from that bound with homocysteine. 0,Binding ofhomocysteine-treated Lp(a) to plasmin-modified fibrin; o,mean and range of binding of homocysteine-treated Lp(a) to wellcoated with the other proteins. (Right) Effect of homocysteine onbinding of Glu-plasminogen to fibrin. Glu-plasminogen was incu-bated 2 hr with 2 mM homocysteine and then added to plasmin-treated fibrin (e) or to untreated fibrin (A). Plasminogen in buffer wasadded to plasmin-treated fibrin (o) or to untreated fibrin (A). Plas-minogen binding was determined by ELISA.

was no difference in the protein elution pattern betweentreated and untreated Lp(a) (Fig. 5). Fibrin binding activityalso appeared in identical fractions; however, the activitywas -10-fold greater with the homocysteine-treated Lp(a).Immunoblot analysis of homocysteine-treated Lp(a) docu-mented the fact that a portion of the Lp(a) particle wasreduced by this compound (Fig. 6). Comparison of nativeLp(a) (lane 1) with Lp(a) treated with 50 mM dithiothreitol(lane 2) shows complete reduction of Lp(a) by dithiothreitol.

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FIG. 4. Effect of sulfhydryl or sulfur compounds on Lp(a) bindingto plasmin-modified fibrin. Lp(a) in buffer mixed with the followingcompounds was added to plasmin-modified fibrin for 90 min, andbinding was determined by ELISA. Lp(a) in 1 mM dithiothreitol (0)or the following at 2 mM each: homocysteine (o), cysteine (*),N-acetylcysteine (A), glutathione (A). Lp(a) was also added in bufferor in the following at 2 mM each: methionine, S-adenosylhomocys-teine, homocystine, or oxidized dithiothreitol. Binding curves forLp(a) in buffer or one ofthe other agents were similar and are shownas the mean (m) ± SD.

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FIG. 5. Gel-filtration chromatography of homocysteine (HC)-treated Lp(a). Lp(a) (0.6 mg) treated overnight with 2 mM homo-cysteine, Lp(a) in buffer, or LDL was applied to a column (1.6 x 54cm) containing Sephacryl S400 high-resolution gel (Pharmacia); 1-mlfractions were collected. Fibrin binding activity was measured onplasmin-treated fibrin plates by ELISA and is expressed by referenceto a Lp(a) binding curve: Lp(a) protein (-), LDL protein ( ),homocysteine-treated Lp(a) fibrin binding activity (n), Lp(a) fibrinbinding activity (A).

Neither Lp(a) protein, as shown by stain, nor immunologicreactivity remained near the top of the slab gel. Apo B-100and apo(a) immunoreactive bands were identified with fasterelectrophoretic mobility than the intact Lp(a), consistentwith the electrophoretic migration of free apolipoproteins.Homocysteine caused a concentration-dependent reduc-

tion of Lp(a) with partial loss of material at the position ofintact Lp(a) and the appearance of free apolipoproteins (Fig.6, lanes 3 and 4). The electrophoretic mobility of the apo(a)bands produced by homocysteine (Fig. 6 Right, lanes 3 and4) was faster than that of the dithiothreitol-derived apo(a)(Fig. 6 Right, lane 2). The lowest concentration of homocys-teine used for these studies, 0.1 mM, also produced resultsqualitatively similar to those shown for 0.5 and 2 mM. Sincedisulfide bond cleavage in proteins is associated with slowerelectrophoretic mobility in SDS/PAGE, these findings sug-gest that the concentrations of homocysteine used producedsignificantly less cleavage of intramolecular disulfide bondsin the apo(a) molecule than that caused by dithiothreitol.

DISCUSSIONThese studies demonstrate the remarkable susceptibility ofLp(a) to sulfhydryl compounds of physiological significance,

protein stain

zf. "o, 'T si

.f.:s I :; '! .-;i Tf.'N

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1 2 3 4

FIG. 6. Western blot analysis of homocysteine-treated Lp(a).Lp(a) (lane 1) treated with 50 mM dithiothreitol (lane 2), 0.5 mMhomocysteine (lane 3), or 2 mM homocysteine (lane 4) for 90 min at37C followed by incubation with iodoacetamide (final concentra-tion, 10 mM) for 30 min was electrophoresed on a 3-8% SDS/polyacrylamide gel followed by electrotransfer to poly(vinylidenedifluoride) membranes. Apolipoproteins were detected with poly-clonal goat anti-apoB-100 (Middle) or with rabbit anti-apo(a) (Right).Gel was stained with Coomassie blue (Left).

including homocysteine, glutathione, and cysteine. In thepresence of these compounds, the binding of Lp(a) to fibrinis significantly enhanced. Homocysteine is an amino acid ofespecial interest with regard to the development of athero-sclerosis. Individuals with inherited homocysteinuria havegreatly elevated blood levels ofhomocysteine. These patientssuccumb in adolescence with prematurely severe atheroscle-rotic vascular and thromboembolic disease (31). More mod-erate elevations in blood homocysteine appear also to beassociated with premature atherosclerosis (31-33). Elevatedlevels of homocysteine have been found in 20-30% of indi-viduals so far studied with premature coronary heart disease(32, 33), peripheral vascular disease (33-36), and stroke (33,34, 37). The mechanisms responsible for atherosclerosis inhomocysteinemia are not clear. Prior studies have shown thathomocysteine can induce endothelial cell injury (38-42).Homocysteine can also enhance autooxidation of LDL cho-lesterol (43, 44) and affect platelet and endothelial cellcoagulant function (38, 45-49). That homocysteine also en-hances binding of Lp(a) to plasmin-degraded fibrin providesa mechanism that relates this metabolic defect to atheroscle-rosis. The concentrations of homocysteine shown in thepresent study to affect fibrin binding ofLp(a), as low as 8 IM,are found as free homocysteine in plasma in vivo (33) and are1-2 orders of magnitude lower than the concentrations ofhomocysteine reported for affecting vascular endothelial celland platelet function. These studies demonstrate that thebinding of Lp(a) to plasmin-modified fibrin is dependent onboth homocysteine and Lp(a) concentration, establishing apossible relationship between these atherogenic agents.The increase in binding induced by homocysteine appears

to be dependent on lysine binding sites in Lp(a), since eACAinhibits homocysteine-induced binding. Evidence for partic-ipation of the apo(a) portion of the Lp(a) particle is providedby the failure ofhomocysteine to induce binding ofLp(a-) orof LDL to the immobilized fibrin surface. The binding ofhomocysteine-treated Lp(a) to plasmin-modified fibrin wasfound to be specific since homocysteine did not increase thebinding of Lp(a) to other immobilized proteins of differentmolecular size and biological activities.Other sulfhydryl compounds also enhance the Lp(a)-fibrin

interaction, including glutathione, which is found in highconcentrations in erythrocytes and platelets (50). The re-quirement of a free sulfhydryl in compounds that enhancebinding to fibrin is evident from our studies that homocys-teine-related sulfur-containing compounds such as methio-nine, S-adenosylhomocysteine, homocystine, as well as ox-idized dithiothreitol did not increase Lp(a) binding. Thehomocysteine effect was specific for Lp(a) as this agent didnot influence the binding of Glu-plasminogen to the fibrinsurface. We have attempted to identify structural changesthat homocysteine may have induced in the Lp(a) particle.Gel-filtration chromatography showed identical coelution ofhomocysteine-treated and untreated Lp(a). However, thefractions from the treated Lp(a) demonstrated greatly in-creased binding to fibrin, indicating that the Lp(a) had beenmodified even though the elution pattern of the Lp(a) particlewas not significantly changed by homocysteine. These stud-ies suggest that the apo(a) remained associated with the LDLparticle under nondenaturing conditions. Western blot anal-ysis of Lp(a) documented that homocysteine caused a con-centration-dependent decrease in the amount of Lp(a) mi-grating at the position of the intact particle, and an increasein free apoB-100 and apo(a). This indicated that homocys-teine had reduced the putative disulfide bond between apoB-100 and apo(a) (4). There were, however, significant differ-ences with respect to the electrophoretic mobility of apo(a)released by homocysteine as opposed to that released bydithiothreitol. Lp(a) reduction by dithiothreitol under condi-tions known to fully reduce most proteins produced an apo(a)

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species of distinctly slower mobility than that produced byhomocysteine. In each instance, however, apo(a) remaineddetectable when probed with an apo(a) antiserum. In prelim-inary immunoblot ligand studies, we have found that fibrindegradation products bind to the free apo(a) produced byhomocysteine, but not to the dithiothreitol reduced molecule,suggesting that reduced apo(a) loses functional activity.The finding of this report may have significance for the

etiology of atherosclerosis. Lp(a) may be altered by homo-cysteine in individuals with hyperhomocysteinemia, by glu-tathione in the microenvironment of the thrombus, or byother reductive mechanisms that remain to be established.Limited reduction may produce a reorientation of apo(a)kringle(s) with exposure of additional binding sites not pre-viously accessible to the fibrin surface. Lp(a) particles withenhanced affinity for fibrin, and perhaps other thrombogenicsurfaces as well, may prove to be an important link betweenthrombosis, atherogenesis, and sulfhydryl compound metab-olism.

Dr. Bruce Gordon (New York Hospital-Cornell Medical Center)provided the Lp(a)-rich plasma. This study was supported in part byU.S. Public Health Service Grant HL-18828 (Specialized Center ofResearch in Thrombosis) and by a grant from the Council forTobacco Research.

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10. Hoff, H. F., Beck, G. J., Skibinsik, C. I., Jurgens, G., O'Neil,J. A., Kramer, J. & Lytle, B. (1988) Circulation 77, 1238-1244.

11. Cushing, G. L., Gaubatz, J. W., Nava, M. L., Burdick, B. J.,Bocan, T. M. A., Guyton, J. R., Weilbaecher, D., DeBakey,M. E., Lawrie, G. M. & Morrisett, J. D. (1989) Arteriosclero-sis 9, 593-603.

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