JBC INTERFERin siRNA transfection reagent

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M305154200 on July 9, 2003

J. Biol. Chem., Vol. 278, Issue 39, 37154-37159, September 26, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/39/37154    most recent
M305154200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsurupa, G.
Right arrow Articles by Medved, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsurupa, G.
Right arrow Articles by Medved, L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Identification and Characterization of Novel Lysine-independent Apolipoprotein(a)-binding Sites in Fibrin(ogen) {alpha}C-domains*

Galina Tsurupa {ddagger} §, Benoît Ho-Tin-Noé § ¶, Eduardo Anglés-Cano ¶ and Leonid Medved {ddagger} ||

From the {ddagger}Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855 and INSERM, U460, Centre Hospitalier Universitaire Bichat-Claude Bernard, 75877 Paris 18, France

Received for publication, May 16, 2003 , and in revised form, July 3, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Accumulation of lipoprotein(a) (Lp(a)) in atherosclerotic plaques is mediated through interaction of fibrin-(ogen) deposits with the apolipoprotein(a) (apo(a)) moiety of Lp(a). It was suggested that because apo(a) competes with plasminogen for binding to fibrin, causing inhibition of fibrinolysis, it could also promote atherothrombosis. Because the fibrin(ogen) {alpha}C-domains bind plasminogen and tissue-type plasminogen activator with high affinity in a Lys-dependent manner, we hypothesized that they could also bind apo(a). To test this hypothesis, we studied the interaction between the recombinant apo(a) A10 isoform and the recombinant {alpha}C-fragment (A{alpha}-(221–610)) corresponding to the {alpha}C-domain by enzyme-linked immunosorbent assay and surface plasmon resonance. Both methods revealed a high affinity interaction (Kd = 19–21 nM) between the immobilized {alpha}C-fragment and apo(a), indicating that the former contains an apo(a)-binding site. This affinity was comparable to that of apo(a) for fibrin. At the same time, no interaction was observed between soluble fibrinogen and immobilized apo(a), suggesting that, in the former, this and other apo(a)-binding sites are cryptic. Further experiments with truncated recombinant variants of the {alpha}C-fragment allowed localization of the apo(a)-binding site to the A{alpha}-(392–610) region. The presence of {epsilon}-aminocaproic acid only slightly inhibited binding of apo(a) to the {alpha}C-fragment, indicating the Lys-independent nature of their interaction. In agreement, the influence of plasminogen or tissue-type plasminogen activator on binding of apo(a) to the {alpha}C-fragment was minimal. These results indicate that the {alpha}C-domains contain novel high affinity apo(a)-binding sites that may provide a Lys-independent mechanism for bringing Lp(a) to places of fibrin deposition such as injured vessels or atherosclerotic lesions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Elevated plasma levels of lipoprotein(a) (Lp(a))1 and fibrinogen are independent risk factors for atherosclerotic cardiovascular diseases (1–3). Numerous in vivo experiments with transgenic animals directly proved involvement of Lp(a) and fibrinogen in the development and progression of atherosclerosis. It was demonstrated that transgenic mice expressing human apolipoprotein(a) (apo(a)), a protein component of Lp(a), are more susceptible to diet-induced atherosclerosis (4–6). It was also shown that in apo(a)-transgenic rabbits, in which apo(a) is efficiently assembled into Lp(a), the latter substantially increases the development of aortic and coronary atherosclerosis and accelerates formation of advanced atherosclerotic lesions (7–9). Finally, it was found that fibrinogen deficiency in apo(a) transgenic mice reduces accumulation of apo(a) in the vessel walls and lesion development, suggesting that fibrin(ogen) may provide one of the major sites to which apo(a) binds to the vessel wall and participates in the generation of atherosclerosis (10).

Although the mechanism through which Lp(a) and fibrin-(ogen) may contribute to the atherogenic processes is still not clearly understood, it seems to be connected with their structure and their ability to interact with each other. Lp(a) is a lipoprotein particle composed of a lipid core and two disulfide-linked apolipoproteins, apoB-100 and apo(a). The lipid core and apoB-100 are shared with low density lipoprotein, the major transporter of cholesterol in human plasma; at the same time, apo(a), which shows a high degree of homology to plasminogen, confers unique properties on Lp(a) (11, 12). Because of these structural similarities, Lp(a) was implicated in the delivery of cholesterol to injured blood vessels (13) and in competition with plasminogen for binding to fibrin and cellular surfaces (14); interaction between Lp(a) and fibrin may play a critical role in both cases. It has been established that both plasminogen and apo(a) contain Lys-binding sites (15, 16). It is also known that binding of plasminogen to fibrin via these sites is important for its conversion into an active enzyme, plasmin (17–19). Numerous studies have demonstrated that Lp(a) and apo(a) also interact with fibrin(ogen) via their Lys-binding sites and compete effectively with plasminogen for its interaction with fibrin (14, 20–24). It was suggested that such competition inhibits generation of active plasmin on fibrin associated with atherosclerotic lesions, resulting in inhibition of fibrinolysis and thereby promoting atherogenesis.

Apo(a) is a one-chain multidomain glycoprotein consisting of a number of kringle domains and a serine protease domain, which are highly homologous to the corresponding domains of plasminogen (11, 12). The COOH-terminal protease-like domain of apo(a) shares 94% sequence homology with that of plasminogen and contains the same catalytic triad; however, the Arg-Val activation cleavage site present in plasminogen is replaced with Ser-Ile in apo(a) (12). The kringle domain of apo(a) adjacent to the protease-like domain is highly homologous to plasminogen kringle domain V, whereas the remaining multiple kringle domains share 61–75% homology with plasminogen kringle domain IV. There are 10 different types of kringle domain IV, one of which appears in a variable number generating isoforms of apo(a) of different sizes (25). Kringle domain IV type 10 of apo(a) contains a Lys-binding site, which may be responsible for its interaction with fibrin(ogen) (26).

Fibrinogen is also a multidomain glycoprotein. It consists of two identical subunits, each of which is formed by three polypeptide chains, A{alpha}, B{beta}, and {gamma} (27). Both the subunits and the chains are linked together by disulfide bonds and form a number of distinct independently folded domains grouped into several structural regions, the central E-region, two identical terminal D-regions, and the {alpha}C-domains (27–30). Each D-region is formed by the COOH-terminal portions of the B{beta}- and {gamma}-chains and a middle portion of the A{alpha}-chain, whereas each {alpha}C-domain is formed by the COOH-terminal portion of the A{alpha}-chain (A{alpha}-(221–610)) (27, 30, 31). Both the D-regions and the {alpha}C-domains contain Lys-dependent plasminogen-binding sites (32–34), which may potentially be involved in the interaction with apo(a) kringle domains. The existence of Lys-dependent apo(a)-binding sites in the plasminogen-binding D-regions has been demonstrated recently using the yeast two-hybrid system (35); nothing is known about the involvement of the {alpha}C-domains in interaction with apo(a).

Because the {alpha}C-domains bind plasminogen and tissue-type plasminogen activator (tPA) in a Lys-dependent manner and with high affinity, which is comparable to that for the interaction between apo(a) and fibrin (22, 33), we hypothesized that they could also bind apo(a) and that the latter could compete for binding with plasminogen and possibly tPA. To test this hypothesis, we examined the interaction between the recombinant apo(a) A10 isoform and the recombinant {alpha}C-domain. We also examined the way in which that interaction is influenced by plasminogen, tPA, and {epsilon}-aminocaproic acid ({epsilon}-ACA), a synthetic analog of Lys. We found that the {alpha}C-domain bound apo(a) with high affinity; however, this binding was inhibited only slightly by excess plasminogen, tPA, or {epsilon}-ACA, indicating that it occurs mainly through a novel Lys-independent binding site. This site was further localized to the COOH-terminal half of the {alpha}C-domain (residues 392–610).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Proteins—Plasminogen-depleted human fibrinogen was purchased from Calbiochem. It also did not contain factor XIII because its incubation with thrombin resulted in non-cross-linked fibrin as revealed by SDS-PAGE under reduced conditions. Recombinant single chain tPA was a Genentech product. Human Glu-plasminogen (form II) was prepared from citrated human plasma by affinity chromatography on Lys-Sepharose 4B (36) and further purified by size exclusion chromatography on Superdex 200 (Amersham Biosciences). Bovine {alpha}-thrombin was from Sigma, and bovine serum albumin was from Calbiochem.

Antibodies—The sheep anti-tPA and goat anti-plasminogen polyclonal antibodies were purchased from Chemicon International, Inc. The peroxidase-conjugated anti-sheep and anti-goat polyclonal antibodies were from Sigma. The sheep anti-apo(a) antibodies and monoclonal antibody A10.2 directed against the lysine-binding site of kringle domain IV type 10 of apo(a) were obtained as described (37, 38).

Expression and Purification of Recombinant Proteins—The recombinant {alpha}C-fragments corresponding to the human fibrinogen {alpha}C-domain (A{alpha}-(221–610)) and its NH2- and COOH-terminal halves (A{alpha}-(221–391) and A{alpha}-(392–610), respectively) were produced in Escherichia coli using the pET-20b expression vector as described earlier (33). They were purified and then refolded according to the procedures described (31, 33).

Recombinant apo(a) A10 was produced in adenovirus-transformed human embryonic kidney cells stably transfected with the pCMV-A10 expression plasmid and cultured in a hollow fiber bioreactor as described (39). The culture medium was supplemented with serine protease inhibitors (20 kallikrein-inhibitory units/ml aprotinin, 1 mmol/liter aminoethylbenzenesulfonyl fluoride, 1 µM D-Phe-Pro-Arg-chloromethyl ketone, 2 mmol/liter EDTA, and 0.01% (w/v) NaN3, final concentrations) and was used for the isolation of recombinant apo(a) by affinity chromatography on Lys-Sepharose 4B equilibrated with 20 mM phosphate buffer (pH 7.4) supplemented with 0.5 M NaCl and serine protease inhibitors. After sample application and washing, the column was equilibrated with 50 mM phosphate buffer (pH 7.4) containing 80 mM NaCl and serine protease inhibitors. To isolate human recombinant apo(a) from adsorbed fetal calf plasminogen present in the culture medium, a stepwise elution procedure was used. Plasminogen was first eluted with 2mM {epsilon}-ACA. The column was then washed, and recombinant apo(a) was eluted with 20 mM {epsilon}-ACA in the same buffer. Fractions containing recombinant apo(a) were pooled, concentrated on dried polyethylene glycol 20,000 (Serva), and dialyzed against 50 mM phosphate buffer containing 80 mM NaCl and 2 mmol/liter EDTA. The recombinant apo(a) preparation was >99% pure as assessed by SDS-PAGE and NH2-terminal sequence analysis using an Applied Biosystems microsequenator equipped with a Model 610A data analysis system. Purified recombinant apo(a) A10 consisted of a serine protease-like region, kringle domain V, and nine kringle domain IV repeats representing each of the kringle domain IV types (type 1 and types 3–10) except type 2.

Solid-phase Binding Assay—Solid-phase binding was performed in plastic microtiter plates using an enzyme-linked immunosorbent assay (ELISA). Microtiter plate wells (Fisher) were coated overnight with 100 µl/well of 10 µg/ml fibrinogen, {alpha}C-fragment, or its truncated variants in 0.1 M Na2CO3 (pH 9.5). To convert fibrinogen into fibrin, the wells with adsorbed fibrinogen were treated with 100 µl/well of a mixture of thrombin (1 NIH unit/ml) and aprotinin (400 units/ml) at 37 °C for 1 h as described previously (40). The wells were then blocked with 1% bovine serum albumin in phosphate-buffered saline (0.02 M sodium phosphate buffer (pH 7.4) and 0.15 M NaCl). Following washing with phosphate-buffered saline containing 0.02% Tween 20, the indicated concentrations of apo(a) in the same buffer were added to the wells and incubated for 1 h. Bound apo(a) was measured by the reaction with the sheep anti-apo(a) polyclonal antibody and the peroxidase-conjugated anti-sheep polyclonal antibody. A 3,3',5,5'-tetramethylbenzidine microwell peroxide substrate (Kirkegaard & Perry Laboratories Inc.) was added to the wells, and the amount of bound ligand was measured spectrophotometrically at 450 nm. Data were analyzed by nonlinear regression analysis using Equation 1,

(Eq. 1)
where A represents absorbance of the oxidized substrate, which is assumed to be proportional to the amount of ligand bound; Amax is the absorption at saturation; [L] is the molar concentration of ligand; and Kd is the dissociation constant.

Biosensor Assay—The interaction of apo(a) with the {alpha}C-fragment and its variants was studied by surface plasmon resonance (SPR) using the IAsys biosensor (Fisons, Cambridge, UK), which measures association/dissociation of proteins in real time (41). The {alpha}C-fragment or its variants were covalently coupled to the aminosilane surface of the cuvette using the glutaraldehyde cross-linking chemistry recommended by the manufacturer. Binding experiments were performed in phosphate-buffered saline containing 0.1 mM phenylmethylsulfonyl fluoride and 0.02% Tween 20 (binding buffer). The association between the immobilized fragments and the added proteins was monitored as the change in the SPR response. The dissociation of the complex was initiated by substitution with the same buffer lacking ligand and monitored in the same manner. To regenerate the surface, complete dissociation of the complex was achieved by adding 10 mM HCl for 1 min following re-equilibration with binding buffer. The traces of the association processes were recorded, and the data were analyzed using the FASTfitTM kinetics analysis software supplied with the instrument as previously described in detail (42). Briefly, the association curves at each concentration of ligand were fitted to the pseudo first-order equation to derive the observed rate constant, kobs (termed on-rate constant in FASTfit). Then, the concentration dependence of kobs was fitted to Equation 2,

(Eq. 2)
to find the association rate constant (ka) from the slope and the dissociation rate constant (kd) from the intercept. The dissociation equilibrium constant (Kd) was calculated as Kd = kd/ka. The values were examined for self-consistency of the data as described (43).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Interaction of Apo(a) A10 with the {alpha}C-Fragment—To check whether the fibrinogen {alpha}C-domains are involved in binding of apo(a), we studied the interaction between the recombinant apo(a) A10 variant and the recombinant {alpha}C-fragment (A{alpha}-(221–610)) corresponding to the {alpha}C-domain; in control experiments, we tested the interaction between apo(a) and fibrinogen. Prior to binding experiments, all three species (the {alpha}C-fragment, fibrinogen, and fibrin) were treated with carboxypeptidase B according to the protocol described (44) to remove possible COOH-terminal Lys residues that could contribute to binding of apo(a). In ELISA, when increasing concentrations of apo(a) A10 were added to the immobilized {alpha}C-fragment, a dose-dependent binding was observed (Fig. 1). The binding was of high affinity with an apparent Kd of 19 nM (Table I). This Kd was very close to that obtained in the same assay for the interaction between apo(a) A10 and immobilized fibrin (Fig. 1 and Table I). This high affinity interaction was confirmed in SPR experiments, in which apo(a) A10 bound to the immobilized {alpha}C-fragment in a dose-dependent manner with a Kd of 21 nM (Fig. 2 and Table I). All these results indicate that the {alpha}C-fragment (and the {alpha}C-domain) contains a high affinity apo(a)-binding site. It should be noted that when either the immobilized {alpha}C-fragment or fibrin was cross-linked with factor XIIIa, the binding curves obtained by ELISA and the calculated Kd values were similar to those obtained with the non-cross-linked species (data not shown), suggesting that cross-linking does not impact this binding.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 1.
Analysis of binding of apo(a) A10 to the immobilized {alpha}C-fragment and fibrin(ogen) by ELISA. Increasing concentrations of apo(a) A10 were incubated with the {alpha}C-fragment (•), fibrin ({circ}), or fibrinogen (inset) immobilized on microtiter wells. Bound protein was detected with specific polyclonal antibodies against apo(a) as described under "Experimental Procedures." Error bars represent the S.D. of triplicate determinations. The curves represent the best fit of the data to Equation 1 to obtain Kd values.

 

View this table:
[in this window]
[in a new window]
  		TABLE I
Dissociation constants for the interaction of apo(a) A10 with immobilized fibrinogen, fibrin, and the recombinant {alpha}C-fragment and its subfragments determined by ELISA and SPR

Values are means ± S.D. of at least three independent experiments. No binding (NB) was observed with the A{alpha}-(211–391) fragment. ND, not determined.

 


View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2.
Analysis of binding of apo(a) A10 to the immobilized {alpha}C-fragment by SPR. Increasing concentrations of apo(a) A10 were added to the immobilized {alpha}C-fragment, and their association was monitored in real time while registering the resonance signal (response). The concentrations of apo(a) were 6, 12, 25, 50, 100, and 200 nM. The inset shows a plot of the values of kobs determined for each association curve versus ligand concentration to derive ka and kd and thus to determine the dissociation equilibrium constants (Kd) presented in Table I. Here and in Figs. 4 and 6, a signal of 600 arc s corresponds to 1 ng of protein bound per mm2 of sensor chip surface.

 



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 4.
Analysis of binding of apo(a) A10 to the {alpha}C-fragment and its NH2- and COOH-terminal fragments (A{alpha}-(221–392) and A{alpha}-(393–610), respectively) by SPR. The association of apo(a) A10 at 200 nM with the immobilized {alpha}C-fragment (dashed curve) and its subfragments, A{alpha}-(221–391) (dotted curve) and A{alpha}-(392–610) (solid curve), was monitored in real time while registering the resonance signal. The inset shows dose-dependent binding of apo(a) A10 to the immobilized A{alpha}-(392–610) fragment; the concentrations of apo(a) A10 were 6, 12, 25, 50, 100, and 200 nM.

 


View larger version (17K):
[in this window]
[in a new window]
  		FIG. 6.
ELISA-detected influence of {epsilon}-ACA and monoclonal antibody A10.2 on the interaction between apo(a) A10 and the immobilized {alpha}C-fragment. A, 0.1 µM apo(a) A10 in the absence (white bar) or presence of 100 mM {epsilon}-ACA (black bar) or 0.2 µM monoclonal antibody A10.2 (mAb; cross-hatched bar), which blocks the lysine-binding function of apo(a) A10, was incubated with microtiter wells coated with the {alpha}C-fragment. Bound protein was detected with specific polyclonal antibodies against apo(a). Error bars represent the S.D. of three independent determinations. B, 0.1 µM apo(a) A10 in the presence (solid curve) or absence (dashed curve) of 100 mM {epsilon}-ACA was added to the immobilized {alpha}C-fragment, and its association was monitored in real time while registering the resonance signal.

 
Interaction of Apo(a) A10 with Fibrinogen—It is well established that the tPA- and plasminogen-binding sites of the {alpha}C-domains and the D-regions are cryptic in fibrinogen and are exposed upon its conversion to fibrin or upon its adsorption to a surface (34, 45). To test whether the apo(a)-binding sites in the fibrinogen {alpha}C-domains are also cryptic, we studied the interaction of soluble fibrinogen with immobilized apo(a) A10. In ELISA, when soluble fibrinogen at a high concentration (1 µM) was added to immobilized apo(a) A10, no binding was observed (data not shown), suggesting that the apo(a)-binding sites in fibrinogen (and in its {alpha}C-domains) are cryptic. It should be mentioned that, in a reverse ELISA experiment, soluble apo(a) A10 bound to immobilized fibrinogen in a dose-dependent manner, although with much lower affinity (Kd = 138 nM) (Fig. 1, inset). However, this binding can be explained by the well established fact that, upon immobilization, fibrinogen undergoes conformational changes resulting in exposure of some fibrin-specific epitopes and binding sites, including those for plasminogen and tPA (45–47). The cryptic character of the apo(a)-binding sites in fibrinogen was confirmed by SPR, in which fibrinogen at the same concentration (1 µM) also exhibited no binding to immobilized apo(a) (data not shown). Thus, the above results indicate that apo(a)-binding sites are cryptic in fibrinogen and become exposed upon its immobilization or conversion into fibrin.

Further Localization of the Apo(a)-binding Site to the {alpha}C-Domain—To further localize the apo(a)-binding site to the {alpha}C-domain, we tested binding of apo(a) A10 to the truncated recombinant variants of the {alpha}C-domain, the A{alpha}-(221–391) and A{alpha}-(392–610) fragments, corresponding to its NH2- and COOH-terminal halves, respectively. In ELISA, apo(a) A10 bound to the immobilized A{alpha}-(392–610) fragment in a dose-dependent manner, whereas practically no interaction was observed with the immobilized A{alpha}-(221–391) fragment (Fig. 3). This binding occurred with a Kd of 22 nM, very close to that determined for the full-length {alpha}C-fragment (Table I). This was confirmed in SPR experiments, in which apo(a) A10 bound to the immobilized COOH-terminal region of the {alpha}C-domain in a dose-dependent manner with a very similar affinity (Kd = 24 nM) (Fig. 4 and Table I). These results indicate that the apo(a)-binding site is located in the COOH-terminal half of the {alpha}C-domain (A{alpha}-(392–610)), the same region that also binds plasminogen and tPA (33). This finding is in agreement with the above-mentioned hypothesis that apo(a) A10 could compete with plasminogen (and/or tPA) for its binding sites in the {alpha}C-domain.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 3.
Analysis of binding of apo(a) A10 to the {alpha}C-fragment and its NH2- and COOH-terminal fragments (A{alpha}-(221–392) and A{alpha}-(393–610), respectively) by ELISA. Increasing concentrations of apo(a) A10 were incubated with microtiter wells coated with the {alpha}C-fragment (•), the A{alpha}-(221–391) fragment ({blacktriangledown}), or the A{alpha}-(392–610) fragment ({diamondsuit}). Bound protein was detected with specific polyclonal antibodies against apo(a) as described under "Experimental Procedures." Error bars represent the S.D. of triplicate determinations. The curves represent the best fit of the data to Equation 1 to obtain the Kd values presented in Table I.

 

Further Characterization of the Interaction between Apo(a) A10 and the {alpha}C-Domain—To check whether there is a competition between apo(a) and plasminogen (and/or tPA) for interaction with the {alpha}C-domains of fibrin, we tested the interaction between the {alpha}C-fragment and apo(a) in the presence and absence of an excess amount of plasminogen or tPA. In ELISA, when apo(a) A10 at 0.1 µM was added to the immobilized {alpha}C-fragment in the presence of a 10-fold molar excess of plasminogen or tPA, the binding was reduced by only 9–13% (Fig. 5A). In another ELISA experiment, when plasminogen or tPA at 0.1 µM was added to the immobilized {alpha}C-fragment, binding in the presence of an 8-fold excess of apo(a) A10 was also reduced only slightly, by 8–15% (Fig. 5, B and C). These results strongly suggest that apo(a) A10, plasminogen, and tPA bind to the {alpha}C-domain mainly via independent binding sites and that neither plasminogen nor tPA binds in fluid phase to apo(a) A10 in a way that would block its interaction with the {alpha}C-domains. To further test this suggestion, the following experiments were performed.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 5.
ELISA-detected competition between apo(a), plasminogen, and tPA for binding to the immobilized {alpha}C-fragment. A, 0.1 µM apo(a) in the absence (white bar) or presence of plasminogen (Pg; black bar) or tPA (cross-hatched bar) at saturating concentrations (1 µM each) was incubated with microtiter wells coated with the {alpha}C-fragment. Bound protein was detected with specific polyclonal antibodies against apo(a). B, plasminogen at 0.1 µM in the absence (white bar) or presence (black bar) of 0.8 µM apo(a) was incubated with microtiter wells coated with the {alpha}C-fragment. Bound protein was detected with specific polyclonal antibodies against plasminogen. C, tPA at 0.1 µM in the absence (white bar) or presence (black bar) of 0.8 µM apo(a) was incubated with microtiter wells coated with the {alpha}C-fragment. Bound protein was detected with specific polyclonal antibodies against tPA. Error bars in all panels represent the S.D. of three independent determinations.

 

Because plasminogen and tPA interact with the {alpha}C-domain in a Lys-dependent manner (38), we further tested the influence of {epsilon}-ACA on the interaction of the latter with apo(a). In ELISA, addition of 100 mM {epsilon}-ACA did not prevent binding of apo(a) to the immobilized {alpha}C-fragment; the binding was reduced by only 8% at this high concentration of {epsilon}-ACA (Fig. 6A). This indicates that apo(a) binds to the {alpha}C-domain mainly in a Lys-independent manner. The Lys-independent character of this binding was confirmed by SPR, in which addition of 100 mM {epsilon}-ACA resulted in only ~10% reduction of the response signal (Fig. 6B). In agreement, this binding was not inhibited by monoclonal antibody A10.2 (Fig. 6A), which blocks strong Lys-binding site(s) of apo(a) (38). Taken together, these results indicate that although apo(a) exhibits some competition with plasminogen and tPA for their specific Lys-dependent binding sites in the {alpha}C-domains, it interacts with the latter mainly through a novel Lys-independent site. Taking into account that this Lys-independent interaction is of high affinity and that there are two {alpha}C-domains in each fibrinogen molecule, one could expect them to contribute substantially to the overall interaction between apo(a) and fibrin.

Lys-independent Interaction of Apo(a) A10 with Fibrin—To evaluate the relative contribution of the Lys-independent interaction to the overall interaction between apo(a) and fibrin, we examined binding of apo(a) to fibrin in the absence and presence of {epsilon}-ACA by ELISA. When increasing concentrations of apo(a) A10 were incubated with immobilized fibrin, in both cases, there was a dose-dependent increase in binding that approached saturation (Fig. 7). The level of binding in the presence of {epsilon}-ACA suggests that the Lys-independent binding accounts for about half of the overall binding. The apparent Kd values for both total and Lys-independent binding were very similar (16 and 15 nM, respectively). Similar results were obtained when fibrin was cross-linked with factor XIIIa (data not shown). The affinity of the Lys-independent binding was also comparable with that determined here for the interaction between apo(a) and the {alpha}C-fragment, suggesting that, in fibrin, this binding may be mediated exclusively by the {alpha}C-domains.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 7.
ELISA-detected influence of {epsilon}-ACA on the interaction between apo(a) A10 and immobilized fibrin. Increasing concentrations of apo(a) A10 in the absence ({circ}) or presence (•) of 100 mM {epsilon}-ACA were incubated with microtiter wells coated with immobilized fibrin, and bound protein was detected with specific polyclonal antibodies against apo(a) as described under "Experimental Procedures." Error bars represent the S.D. of triplicate determinations. The curves represent the best fit of the data to Equation 1 to obtain Kd values. The Kd values in the presence and absence of {epsilon}-ACA were found to be 15 ± 1.7 and 16 ± 1.5 nM, respectively.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Because fibrin(ogen) plays a prominent role in accumulation of Lp(a) in the vessel walls and development of atherosclerosis (10), an understanding of the molecular mechanisms underlying this pathological process requires a better knowledge of the mechanism of interactions between these two plasma components. Numerous studies indicate that such an interaction is Lys-dependent and occurs through Lys-binding sites of the apo(a) component of Lp(a) and complementary Lys-containing sites on fibrin(ogen) that are also involved in binding of plasminogen (14, 20–24). The present study was initiated to examine whether the fibrinogen {alpha}C-domains, which include high affinity Lys-containing binding sites for plasminogen and tPA (33), would also bind apo(a) through these sites. The results obtained revealed that apo(a) indeed interacts with the {alpha}C-domains with high affinity; however, it turned out that this interaction requires participation of novel Lys-independent apo(a)-binding sites that are distinct from those for plasminogen and tPA.

The most popular models for the study of interaction of apo(a) with fibrin(ogen) include immobilized intact or plasmin-treated fibrinogen or fibrin (20, 22, 48–51). Both immobilized species bind Lp(a) or apo(a); however, the plasmin-treated one exhibits much higher binding, which could be explained by the presence of plasmin-generated COOH-terminal Lys residues (14, 20–24). At the same time, it was not clear whether fibrinogen interacts with apo(a) in solution. Our study clearly demonstrates that the apo(a)-binding site in the {alpha}C-domain and the apo(a)-binding sites in other fibrin(ogen) regions are cryptic in soluble fibrinogen and are exposed in fibrin. They are also partially exposed in immobilized fibrinogen, which exhibited lower affinity for apo(a) than immobilized fibrin. These data suggest that Lp(a) does not interact with fibrinogen in the circulation, but should interact strongly with fibrin deposited on vessel walls and atherosclerotic lesions. A similar situation was observed with plasminogen and tPA (33), fibronectin (40), and Mac-1 (52), each of which interacted only with fibrin or immobilized fibrinogen. Thus, this study reinforces previous findings that fibrinogen is inert in the circulation and becomes reactive upon its conversion to fibrin. Such reactivity brings various proteins and cell types to places of fibrin deposition and provides their participation in subsequent fibrin-dependent physiological and pathological processes, including atherosclerosis.

Our study revealed that binding of apo(a) to the {alpha}C-domains is mainly Lys-independent because {epsilon}-ACA decreased this binding by only 8–15% (Figs. 5 and 6). Although, according to the current view, the interaction between apo(a) and fibrin(ogen) is regarded to be mainly Lys-dependent, the Lys-independent interaction was also noticed in a number of previous studies; however, it was either ignored or regarded as "nonspecific" and was subtracted from the overall interaction (20, 22, 48, 50). At the same time, our experiments with apo(a) and fibrin revealed that this interaction is substantial and may account for almost half of the overall interaction between these two components (Fig. 7). This is in agreement with a previous report showing that addition of {epsilon}-ACA results in only an ~2-fold drop in binding of Lp(a) to immobilized fibrinogen (20). Furthermore, it was reported recently that a high concentration of {epsilon}-ACA inhibits binding of a recombinant apo(a) fragment (kringle domain V plus a protease-like domain) to plasmin-treated fibrinogen by only 65% and was suggested that Lp(a) interacts with the latter via both Lys-dependent and Lys-independent binding sites (51). Our study further proved the presence of Lys-independent binding sites in fibrin(ogen) and localized them to its {alpha}C-domains, viz. in the COOH-terminal regions of these domains between A{alpha}-chain residues 392 and 610.

Although the {alpha}C-domain contains a high affinity Lys-dependent plasminogen-binding site, its interaction with apo(a), which contains both Lys-dependent and Lys-independent sites, occurs almost exclusively through the latter ones, i.e. apo(a) does not compete well with plasminogen for its binding site in the {alpha}C-domain. A similar situation may occur with the other Lys-dependent plasminogen-binding site, which is located in the D-region and includes A{alpha}-(148–160) (32). Indeed, it was shown recently using the yeast two-hybrid system that the D-region contains specific apo(a)-binding sites in residues 207–235 of its {gamma}-chain and in the corresponding homologous portion of its {beta}-chain (35). However, these sites do not overlap with A{alpha}-(148–160). This raises the question of whether apo(a) competes for the plasminogen-binding site in the D-region or interacts only with the specific apo(a)-binding sites, similar to the situation found in this study for the {alpha}C-domain. Direct competition experiments with apo(a), plasminogen, and the fibrinogen D-fragment and its subfragments are required to address this question. At this stage, it seems that the COOH-terminal Lys residues generated by plasmin treatment of fibrin(ogen) are the only well characterized Lys-dependent binding sites for which apo(a) competes with plasminogen (23, 24, 53).

Competition between the apo(a) component of Lp(a) and plasmin(ogen) for the Lys-containing binding sites in fibrin(ogen), which results in inhibition of plasmin generation and thus modulation of fibrin-associated fibrinolysis, is regarded as the major established mechanism by which both apo(a) and fibrin promote atherogenesis (6, 24). At the same time, this and other studies (35, 51) indicate the presence of the Lys-independent interaction between apo(a) and fibrin. Considering that such an interaction could account for almost half of the overall apo(a) binding, as demonstrated in Fig. 7, and that it occurs with high affinity, one can speculate that it may contribute substantially to the interaction between Lp(a) and fibrin and in fact may represent an alternative Lys-independent mechanism that may promote atherogenesis by bringing Lp(a) to fibrin-containing atherosclerotic lesions. The {alpha}C-domains that provide this interaction may play a major role in this mechanism. One can also speculate that the Lys-independent mechanism may be most important at the early stage of accumulation of Lp(a) in plaque lesions, whereas the Lys-dependent one prevails after fibrin-associated activation of plasminogen into plasmin and subsequent generation of COOH-terminal Lys residues.

In summary, in this study, we identified and characterized novel high affinity apo(a)-binding sites in the fibrin(ogen) {alpha}C-domains that are cryptic in fibrinogen and become exposed upon its immobilization or conversion into fibrin and that could be responsible for as much as half of the overall interaction between Lp(a) and fibrin. This finding indicates that the {alpha}C-domains should contribute substantially to the interaction between fibrin and Lp(a) and thus to the development of atherothrombosis and also suggests that the {alpha}C-domains could be a potential target in the development of substances controlling this process.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HL-56051 (to L. M.) and by an Adrienne et Pierre Sommer grant from the Fondation de France (to E. A.-C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

|| To whom correspondence should be addressed: Jerome H. Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0719; Fax: 301-738-0740; E-mail: medvedL{at}usa.redcross.org.

1 The abbreviations used are: Lp(a), lipoprotein(a); apo(a), apolipoprotein(a); tPA, tissue-type plasminogen activator; {epsilon}-ACA, {epsilon}-aminocaproic acid; ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon resonance. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Kenneth Ingham for helpful discussions and criticism. The technical assistance of Stéphane Loyau is gratefully acknowledged.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Danesh, J., Collins, R., and Peto, R. (2000) Circulation 102, 1082–1085[Abstract/Free Full Text]
  2. Danesh, J., Collins, R., Appleby, P., and Peto, R. (1998) J. Am. Med. Assoc. 279, 1477–1482[Abstract/Free Full Text]
  3. Bini, A., and Kudryk, B. J. (1995) Ann. N. Y. Acad. Sci. 748, 461–471[Medline] [Order article via Infotrieve]
  4. Lawn, R. M., Wade, D. P., Hammer, R. E., Chiesa, G., Verstuyft, J. G., and Rubin, E. M. (1992) Nature 360, 670–672[CrossRef][Medline] [Order article via Infotrieve]
  5. Lawn, R. M., Pearle, A. D., Kunz, L. L., Rubin, E. M., Reckless, J., Metcalfe, J. C., and Grainger, D. J. (1996) J. Biol. Chem. 271, 31367–31371[Abstract/Free Full Text]
  6. Boonmark, N. W., Lou, X. J., Yang, Z. J., Schwartz, K., Zhang, J. L., Rubin, E. M., and Lawn, R. M. (1997) J. Clin. Invest. 100, 558–564[Medline] [Order article via Infotrieve]
  7. Fan, J., Shimoyamada, H., Sun, H., Marcovina, S., Honda, K., and Watanabe, T. (2001) Arterioscler. Thromb. Vasc. Biol. 21, 88–94[Abstract/Free Full Text]
  8. Ichikawa, T., Unoki, H., Sun, H., Shimoyamada, H., Marcovina, S., Shikama, H., Watanabe, T., and Fan, J. (2002) Am. J. Pathol. 160, 227–236[Abstract/Free Full Text]
  9. Sun, H., Unoki, H., Wang, X., Liang, J., Ichikawa, T., Arai, Y., Shiomi, M., Marcovina, S. M., Watanabe, T., and Fan, J. (2002) J. Biol. Chem. 277, 47486–47492[Abstract/Free Full Text]
  10. Lou, X. J., Boonmark, N. W., Horrigan, F. T., Degen, J. L., and Lawn, R. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12591–12595[Abstract/Free Full Text]
  11. Eaton, D. L., Fless, G. M., Kohr, W. J., McLean, J. W., Xu, Q. T., Miller, C. G., Lawn, R. M., and Scanu, A. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3224–3228[Abstract/Free Full Text]
  12. McLean, J. W., Tomlinson, J. E., Kuang, W. J., Eaton, D. L., Chen, E. Y., Fless, G. M., Scanu, A. M., and Lawn, R. M. (1987) Nature 330, 132–137[CrossRef][Medline] [Order article via Infotrieve]
  13. Brown, M. S., and Goldstein, J. L. (1987) Nature 330, 113–114[CrossRef][Medline] [Order article via Infotrieve]
  14. Anglés-Cano, E., de la Pena, D. A., and Loyau, S. (2001) Ann. N. Y. Acad. Sci. 936, 261–275[Abstract/Free Full Text]
  15. Wiman, B., and Wallen, P. (1977) Thromb. Res. 10, 213–222[CrossRef][Medline] [Order article via Infotrieve]
  16. Guevara, J., Jr., Jan, A. Y., Knapp, R., Tulinsky, A., and Morrisett, J. D. (1993) Arterioscler. Thromb. 13, 758–770[Abstract/Free Full Text]
  17. Wiman, B., and Collen, D. (1978) Nature 272, 549–550[CrossRef][Medline] [Order article via Infotrieve]
  18. Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912–2919[Abstract/Free Full Text]
  19. Verheijen, J. H., Nieuwenhuizen, W., and Wijngaards, G. (1982) Thromb. Res. 27, 377–385[CrossRef][Medline] [Order article via Infotrieve]
  20. Harpel, P. C., Gordon, B. R., and Parker, T. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3847–3851[Abstract/Free Full Text]
  21. Loscalzo, J., Weinfeld, M., Fless, G. M., and Scanu, A. M. (1990) Arteriosclerosis 10, 240–245[Abstract/Free Full Text]
  22. Rouy, D., Koschinsky, M. L., Fleury, V., Chapman, J., and Anglés-Cano, E. (1992) Biochemistry 31, 6333–6339[CrossRef][Medline] [Order article via Infotrieve]
  23. Bas, L. C., Duif, P. F., Gimpel, J. A., Kortlandt, W., Bouma, B. N., and van Rijn, H. J. (1992) Thromb. Haemostasis 68, 185–188[Medline] [Order article via Infotrieve]
  24. Hervio, L., Durlach, V., Girard-Globa, A., and Anglés-Cano, E. (1995) Biochemistry 34, 13353–13358[CrossRef][Medline] [Order article via Infotrieve]
  25. Lackner, C., Cohen, J. C., and Hobbs, H. H. (1993) Hum. Mol. Genet. 2, 933–940[Abstract/Free Full Text]
  26. Klezovitch, O., and Scanu, A. M. (1996) Arterioscler. Thromb. Vasc. Biol. 16, 392–398[Abstract/Free Full Text]
  27. Henschen, A., and McDonagh, J. (1986) in Blood Coagulation (Zwaal, R. F. A., and Hemker, H. C., eds) pp. 171–241, Elsevier Science B.V., Amsterdam
  28. Privalov, P. L., and Medved, L. V. (1982) J. Mol. Biol. 159, 665–683[CrossRef][Medline] [Order article via Infotrieve]
  29. Medved, L., Litvinovich, S., Ugarova, T., Matsuka, Y., and Ingham, K. (1997) Biochemistry 36, 4685–4693[CrossRef][Medline] [Order article via Infotrieve]
  30. Weisel, J. W., and Medved, L. (2001) Ann. N. Y. Acad. Sci. 936, 312–327[Abstract/Free Full Text]
  31. Tsurupa, G., Tsonev, L., and Medved, L. (2002) Biochemistry 41, 6449–6459[CrossRef][Medline] [Order article via Infotrieve]
  32. Nieuwenhuizen, W. (2001) Ann. N. Y. Acad. Sci. 936, 237–246[Abstract/Free Full Text]
  33. Tsurupa, G., and Medved, L. (2001) Biochemistry 40, 801–808[CrossRef][Medline] [Order article via Infotrieve]
  34. Medved, L., and Nieuwenhuizen, W. (2003) Thromb. Haemostasis 89, 409–419[Medline] [Order article via Infotrieve]
  35. Klose, R., Fresser, F., Kochl, S., Parson, W., Kapetanopoulos, A., Fruchart-Najib, J., Baier, G., and Utermann, G. (2000) J. Biol. Chem. 275, 38206–38212[Abstract/Free Full Text]
  36. Deutsch, D. G., and Mertz, E. T. (1970) Science 170, 1095–1096[Abstract/Free Full Text]
  37. Kang, C., Dominguez, M., Loyau, S., Miyata, T., Durlach, V., and Anglés-Cano, E. (2002) Arterioscler. Thromb. Vasc. Biol. 22, 1232–1238[Abstract/Free Full Text]
  38. Dominguez, M., Rojas, G., Loyau, S., Bazurco, M., Sorell, L., and Anglés-Cano, E. (2001) Biochim. Biophys. Acta 1548, 72–80[CrossRef][Medline] [Order article via Infotrieve]
  39. Anglés-Cano, E., Loyau, S., Cardoso-Saldana, G., Couderc, R., and Gillery, P. (1999) J. Lipid Res. 40, 354–359[Abstract/Free Full Text]
  40. Makogonenko, E., Tsurupa, G., Ingham, K., and Medved, L. (2002) Biochemistry 41, 7907–7913[CrossRef][Medline] [Order article via Infotrieve]
  41. Johnsson, B., Lofas, S., and Lindquist, G. (1991) Anal. Biochem. 198, 268–277[CrossRef][Medline] [Order article via Infotrieve]
  42. Gorgani, N. N., Parish, C. R., and Altin, J. G. (1999) J. Biol. Chem. 274, 29633–29640[Abstract/Free Full Text]
  43. Schuck, P., and Minton, A. P. (1996) Trends Biochem. Sci. 21, 458–460[CrossRef][Medline] [Order article via Infotrieve]
  44. Ellis, V., and Dano, K. (1993) J. Biol. Chem. 268, 4806–4813[Abstract/Free Full Text]
  45. Medved, L., Tsurupa, G., and Yakovlev, S. (2001) Ann. N. Y. Acad. Sci. 936, 185–204[Abstract/Free Full Text]
  46. Shiba, E., Lindon, J. N., Kushner, L., Matsueda, G. R., Hawiger, J., Kloczewiak, M., Kudryk, B., and Salzman, E. W. (1991) Am. J. Physiol. 260, C965–C974
  47. Moskowitz, K. A., Kudryk, B., and Coller, B. S. (1998) Thromb. Haemostasis 79, 824–831[Medline] [Order article via Infotrieve]
  48. Huby, T., Schroder, W., Doucet, C., Chapman, J., and Thillet, J. (1995) Biochemistry 34, 7385–7393[CrossRef][Medline] [Order article via Infotrieve]
  49. Klezovitch, O., Edelstein, C., and Scanu, A. M. (1996) J. Clin. Invest. 98, 185–191[Medline] [Order article via Infotrieve]
  50. Sangrar, W., and Koschinsky, M. L. (2000) Biochem. Cell Biol. 78, 519–525[CrossRef][Medline] [Order article via Infotrieve]
  51. Xue, S., Madison, E. L., and Miles, L. A. (2001) Thromb. Haemostasis 86, 1229–1237[Medline] [Order article via Infotrieve]
  52. Lishko, V. K., Kudryk, B., Yakubenko, V. P., Yee, V. C., and Ugarova, T. P. (2002) Biochemistry 41, 12942–12951[CrossRef][Medline] [Order article via Infotrieve]
  53. Rouy, D., Laplaud, P. M., Saboureau, M., and Anglés-Cano, E. (1992) Arterioscler. Thromb. 12, 146–154[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Nephrol Dial TransplantHome page
A. Undas, M. Kolarz, G. Kopec, and W. Tracz
Altered fibrin clot properties in patients on long-term haemodialysis: relation to cardiovascular mortality
Nephrol. Dial. Transplant., June 1, 2008; 23(6): 2010 - 2015.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/39/37154    most recent
M305154200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal