Antifibrinolytic effect of single apo(a) kringle domains: relationship to fibrinogen binding

Mona N. Rahman, Vitali Petrounevitch, Zongchao Jia and Marlys L. Koschinsky1,

Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for the development of atherosclerotic disease which may be attributable to the ability of Lp(a) to attenuate fibrinolysis. A generally accepted mechanism for this effect involves direct competition of Lp(a) with plasminogen for fibrin(ogen) binding sites thus reducing the efficiency of plasminogen activation. Efforts to determine the domains of apolipoprotein(a) [apo(a)] which mediate fibrin(ogen) interactions have yielded conflicting results. Thus, the purpose of the present study was to determine the ability of single KIV domains of apo(a) to bind plasmin-treated fibrinogen surfaces as well to determine their effect on fibrinolysis using an in vitro clot lysis assay. A bacterial expression system was utilized to express and purify apo(a) KIV 2 , KIV 7 , KIV 9 {Delta}Cys (which lacks the seventh unpaired cysteine) and KIV 10 which contains a strong lysine binding site. We also expressed and examined three mutant derivatives of KIV 10 to determine the effect of changing critical residues in the lysine binding site of this kringle on both fibrin(ogen) binding and fibrin clot lysis. Our results demonstrate that the strong lysine binding site in apo(a) KIV 10 is capable of mediating interactions with plasmin-modified fibrinogen in a lysine-dependent manner, and that this kringle can increase in vitro fibrin clot lysis time by ~43% at a concentration of 10 µM KIV 10 . The ability of the KIV 10 mutant derivatives to bind plasmin-modified fibrinogen correlated with their lysine binding capacity. Mutation of Trp 70 to Arg abolished binding to both lysine–Sepharose and plasmin-modified fibrinogen, while the Trp 70 ->Phe and Arg 35 ->Lys substitutions each resulted in decreased binding to these substrates. None of the KIV 10 mutant derivatives appeared to affect fibrinolysis. Apo(a) KIV 7 contains a lysine- and proline-sensitive site capable of mediating binding to plasmin-modified fibrinogen, albeit with a lower apparent affinity than apo(a) KIV 10 . However, apo(a) KIV 7 had no effect on fibrinolysis in vitro . Apo(a) KIV 2 and KIV 9 {Delta}Cys did not bind measurably to plasmin-modified fibrinogen surfaces and did not affect fibrinolysis in vitro .

Keywords: apolipoprotein(a)/fibrinogen/fibrinolysis/kringles/lysine binding


    Introduction
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 Abstract
 Introduction
 Materials and methods
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Since its discovery ( Berg, 1963Go ), a number of epidemiological studies of both case-control and prospective design have correlated elevated plasma concentrations of lipoprotein(a) [Lp(a)] with an increased risk for the development of coronary heart disease (CHD) ( Marcovina and Koschinsky, 1998Go ; Marcovina et al., 1999Go ). Within the human population, plasma Lp(a) levels vary over 1000-fold, ranging from less than 0.1 mg dl –1 to over 100 mg dl –1 . The risk of developing CHD appears to more than double at Lp(a) concentrations above an apparent coronary risk threshold of 20–30 mg dl –1 (Rhoads et al., 1986Go ; Durrington et al., 1988Go ).

Lp(a) closely resembles low-density lipoprotein (LDL) in both lipid composition and the presence of apolipoprotein B-100 [apoB-100]. It is distinguished from LDL by the presence of the unique glycoprotein apolipoprotein(a) [apo(a)], which is covalently linked to the apoB-100 moiety of the lipoprotein by a single disulfide bond (Fless et al., 1986Go ; Brunner et al., 1993Go ; Koschinsky et al., 1993Go ) and likely confers the unique structural and functional properties attributed to Lp(a). Human apo(a) bears a striking similarity to plasminogen, a serine protease zymogen involved in the fibrinolytic cascade. Unlike plasminogen, which contains five distinct kringle domains (numbered I–V) followed by a serine protease domain, apo(a) consists of multiple tandemly-repeated motifs which closely resemble plasminogen kringle IV (KIV), followed by sequences which are homologous to the kringle V (KV) and protease domains of plasminogen, respectively (McLean et al., 1987Go ). The protease domain of apo(a) is catalytically inactive ( Gabel and Koschinsky, 1995Go ), but has been implicated in mediating the high affinity interaction between apo(a) and plasminogen (Sangrar et al., 1997Go ). The KIV domains of apo(a) can be classified into 10 types (designated types 1 to 10), based on amino acid sequence (McLean et al., 1987Go ). Each KIV domain is present in single copy with the exception of KIV type 2 (KIV 2 ) which is present in variable number resulting in the considerable size heterogeneity of Lp(a) in the population (van der Hoek et al., 1993Go ; Lackner et al., 1993Go ). A free cysteine residue in KIV 9 (Cys 4057 ) is involved in disulfide linkage to the apoB-100 component of LDL to form covalent Lp(a) particles (Brunner et al., 1993Go ; Koschinsky et al., 1993Go ).

Plasminogen interacts with biological substrates through lysine binding sites (LBS) present within kringle domains I and IV (Lerch et al., 1980Go ; Lucas et al., 1983Go ). Of the KIV domains in apo(a), KIV 10 contains a relatively strong LBS ( K D for {varepsilon}-aminocaproic acid of ~33 µM; M.N.Rahman and M.L.Koschinsky, in preparation ), with all of the residues present which have been implicated in the LBS of plasminogen KIV, the only exception being Arg 35 which corresponds to a lysine at this position in plasminogen (McLean et al., 1987Go ; Guevara et al., 1993Go ; Mikol et al., 1996Go ; Mochalkin et al., 1999Go ). Apo(a) KIV types 5, 6, 7 and 8 contain comparatively weaker LBS (e.g. K D for {varepsilon}-aminocaproic acid of ~230 µM for KIV 7 ; M.N.Rahman and M.L.Koschinsky, in preparation ) which are masked in the context of Lp(a), but are essential in mediating initial non-covalent interactions which precede covalent Lp(a) assembly (Ernst et al., 1995Go ; Gabel et al., 1996Go ). Moreover, non-covalent interactions of KIV 9 with LDL were demonstrated to be sensitive to both phenylalanine and lysine, and modeling studies suggested the presence of overlapping lysine and phenylalanine binding sites within this kringle (Rahman et al., 1998Go ). Based on molecular modeling studies, apo(a) kringle IV types 1 to 4 do not appear to contain functional LBS (Guevara et al., 1993Go ).

Despite extensive studies aimed at understanding the structure and function of apo(a)/Lp(a), the basis for the role of this lipoprotein in atherosclerosis remains unclear. Due to its striking homology to plasminogen coupled with its lack of protease activity, apo(a) has been postulated to interfere with the normal fibrinolytic function of plasminogen in vivo. Indeed, apo(a) has been shown to attenuate fibrin clot lysis both in vitro ( Sangrar et al., 1995Go ), as well as in vivo using a transgenic apo(a) mouse model (Palabrica et al., 1995Go ) and a rabbit jugular vein thrombosis model (Biemond et al., 1997Go ). A commonly accepted mechanism for the antifibrinolytic effect of apo(a) involves displacement of plasminogen and/or tPA from their respective binding sites on fibrin(ogen), thereby interfering with the formation of a ternary complex ( Hoylaerts et al., 1982Go ) which is required for efficient plasminogen activation. Thus, several studies have focused on elucidating the domain(s) of apo(a) responsible for binding intact and plasmin-modified fibrin(ogen). Studies using proteolytically-cleaved Lp(a) demonstrated that this interaction is lysine dependent, and is mediated by sequences within the C-terminal half of apo(a) (i.e. spanning KIV 5 –KIV 10 , followed by the KV and protease-like domains ) (Huby et al., 1995Go ). However, attempts to identify more precisely the domain in apo(a) which mediates its interaction with fibrin(ogen) have proven controversial. For example, Scanu and colleagues have provided evidence that fibrinogen binding by apo(a) involves a lysine- and proline-sensitive site located in the domain spanning apo(a) KIV 5 to KIV 8 (most likely KIV 8 ), but not the LBS in KIV 10 (Klezovitch et al., 1996Go ; Scanu et al., 1997Go ). However, studies using recombinant KIV 10 expressed in Escherichia coli have demonstrated that this domain accounts for the majority, but not the entire, interaction of Lp(a) with a plasmin-treated fibrinogen surface (LoGrasso et al., 1994Go ). This is consistent with a study by Boonmark et al. (1997) which demonstrated that recombinant apo(a) lacking a functional LBS in KIV 10 (due to substitution of both Asp 54 and Asp 56 with Ala ) shows reduced binding to plasmin-modified fibrin [~60% relative to wild-type apo(a)]. Competition studies have also suggested the contribution of sequences within the region spanning KV to the protease domain in mediating this interaction (Xue et al., 1999Go ).

The purpose of the present study was to determine the ability of single KIV domains of apo(a) to bind plasmin-treated fibrinogen surfaces, as well as to study the effects of these kringles on fibrinolysis using an in vitro clot lysis assay. A bacterial expression system was utilized to produce KIV 2 , KIV 7 , KIV 9 {Delta}Cys (which lacks the free cysteine) and KIV 10 . We also expressed and analyzed three mutants of KIV 10 (KIV 10 Trp 70 ->Arg, KIV 10 Trp 70 ->Phe and KIV 10 Arg 35 ->Lys ) to examine the effect of substituting residues of the LBS on lysine-binding, on binding to fibrin(ogen) surfaces, and on fibrinolysis. We demonstrated that both KIV 7 and KIV 10 interact with plasmin-modified fibrinogen in a lysine- and arginine-sensitive manner; interaction of KIV 7 with this surface is also proline-dependent. The lysine-binding capacity of the KIV 10 mutant derivatives was correlated with their ability to bind plasmin-modified fibrinogen. Substitution of the Trp 70 residue of KIV 10 with Arg abolished its ability to bind both lysine–Sepharose as well as plasmin-modified fibrinogen. The more conservative Trp 70 ->Phe substitution as well as the Arg 35 ->Lys substitution reduced the ability of the corresponding derivatives to bind both lysine–Sepharose as well as plasmin-modified fibrinogen. However, only wild-type KIV 10 was observed to attenuate fibrinolysis. Despite their ability to interact with plasmin-modified fibrinogen, neither KIV 7 nor the KIV 10 mutant derivatives had any observable effect on fibrinolysis in vitro .


    Materials and methods
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 Abstract
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 Materials and methods
 Results
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 References
 
Construction of recombinant apo(a) expression plasmids in pET16b

Construction of KIV 9 {Delta}Cys-pET16b has been previously described (Rahman et al., 1998Go ); expression plasmids encoding recombinant KIV 2 , KIV 7 and KIV 10 were constructed in a similar manner. Sequences corresponding to the KIV 2 domain were amplified from the pRK5-SK2 vector (Sangrar et al., 1994Go ) using primers A and D (Table I Go ) . Sequences corresponding to the KIV 7 domain were amplified from the pRK5-SK7 vector (B.R.Gabel, unpublished data) using primers B and D (Table I Go ). Sequences corresponding to the KIV 10 domain were amplified from the pRK5-SK10 vector (Sangrar et al., 1994Go ) using primers C and D (Table I Go ). All primers contain an engineered Nde I site thereby resulting in PCR products flanked by Nde I sites. All PCR products were made blunt-ended with T4 DNA polymerase (New England Biolabs) and cloned into the Eco RV site of pBluescript SK + for DNA sequence analysis. Following digestion of the plasmids with Nde I, resultant fragments encoding each kringle derivative were cloned into the Nde I-digested pET16b vector (Novagen). The resulting recombinant plasmids were designated KIV 2 -pET16b, KIV 7 -pET16b and KIV 10 -pET16b, respectively, and were used to transform the E.coli strain BL21 (DE3) (Novagen) for protein expression as previously described (Rahman et al., 1998Go ).


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Table I. PCR primer sequences
 
Construction of expression plasmids in pET16b encoding the mutant derivatives KIV 10 Trp 70 ->Arg, KIV 10 Trp 70 ->Phe and KIV 10 Arg 35 ->Lys

PCR-based mutagenesis strategies were employed to replace the tryptophan residue at position 70 of KIV 10 with an arginine (KIV 10 Trp 70 ->Arg ) and a phenylalanine (KIV 10 Trp 70 ->Phe ), respectively. For KIV 10 Trp 70 ->Arg, the KIV 10 -pET16b plasmid was used as a template for two separate PCR amplifications using primer pairs C and 1a (Fragment 1; 268 bp) and D and 1b (Fragment 2; 167 bp), respectively (Table I Go ). Both resultant PCR products were made blunt-ended and each cloned into the Eco RV site of pBluescript SK + for DNA sequence analysis. Fragments were then digested with Nde I and Hha I and ligated into the Nde I-restricted pET16b vector in a three-part ligation. The resultant construct was confirmed by DNA sequence analysis and designated KIV 10 Trp 70 ->Arg-pET16b. The KIV 10 Trp 70 ->Phe-pET16b plasmid was constructed in a similar manner. Sequences were amplified using primers C and 2a, and primers D and 2b, resulting in DNA fragments of 262 and 156 bp, respectively (see Table I Go ). The fragments were confirmed by sequence analysis, digested with Nde I and Bst BI and ligated as a three-part ligation into Nde I-digested pET16b.

In order to replace the arginine residue at position 35 of KIV 10 with a lysine (KIV 10 Arg 35 ->Lys ), the coding and signal sequences of KIV 10 in the pRK5-SK10 vector (Sangrar et al., 1994Go ) were amplified by PCR and subcloned into pBluescript SK + that had been digested with Xba I and Sal I. This intermediate vector was utilized as a template for PCR-mediated mutagenesis carried out by the method of Nelson and Long (1989). The mutagenic primer was primer 3a (see Table I Go ). The mutant PCR product (405 bp) was digested with Avr II and Msc I and the resultant fragment was cloned into pRK5-SK10 which had also been digested with these restriction enzymes. The mutant KIV 10 coding sequence was then amplified and cloned into the Nde I site of pET16b using primers C and D as described for the KIV 10 -pET16b plasmid; the final construct was designated KIV 10 Arg 35 ->Lys-pET16b.

Expression and purification of apo(a) kringle IV domains in E.coli

The expression and purification of recombinant kringle IV derivatives was performed essentially as previously described for KIV 9 {Delta}Cys (Rahman et al., 1998Go ). Briefly, 3 l of LB containing 50 µg ml –1 ampicillin were inoculated with an overnight culture of BL21 (DE3) transformed with the respective expression vectors, and incubated with shaking at 37°C until the culture reached an OD 600 of ~0.6. Protein expression was induced by the addition isopropyl-ß- D -thiogalactopyranoside to a final concentration of 1 mM. Cultures were grown for an additional 3 h after induction and subsequently stored at 4°C overnight. Cells were harvested by centrifugation at 5000 g for 10 min at 4°C. The resulting cell pellets were resuspended in a total of 40 ml of binding buffer (20 mM Tris–HCl pH 7.9, 0.5 M NaCl, 5 mM imidazole) and lysed by sonication. The lysate was centrifuged at 20 000 g for 20 min at 4°C. Subsequently, the insoluble fraction was resuspended in 20 ml of binding buffer, sonicated to release trapped proteins and centrifuged at 20 000 g for 15 min as described above. The resulting pellet was resuspended in 20 ml binding buffer containing 6 M guanidine–HCl, 0.5% Triton X-100, 5 mM DTT and incubated with rocking at 4°C for 2–3 days to allow solubilization. The denatured protein was then diluted 5-fold with binding buffer and refolded by incubation at 4°C in the presence of oxidized and reduced glutathione (1.25 mM each) for 1–2 days. Remaining insoluble debris was removed by centrifugation at 39 000 g for 20 min at 4°C.

For purification using Ni 2+ –Sepharose chromatography, HisBind Resin (Novagen) was washed, charged with 50 mM NiSO 4 and equilibrated in binding buffer. The resin was added to the solubilized, refolded crude protein and incubated overnight at 4°C with rocking to allow adsorption of the protein. Following centrifugation at 1000 g , the majority of the supernatant containing unbound protein was removed by aspiration and the resin stacked in a 2.5 ml column. Chromatography over the Ni 2+ –Sepharose column was performed in the presence of reduced and oxidized glutathione (each at a final concentration of 1.25 mM). The column was washed with six column volumes of binding buffer, followed by six column volumes of wash buffer (20 mM Tris–HCl pH 7.9, 0.5 M NaCl, 60 mM imidazole). Specifically-bound protein was eluted from the column with elution buffer (20 mM Tris–HCl pH 7.9, 0.5 M NaCl, 1 M imidazole). Fractions containing the partially-purified protein were pooled for further purification by size exclusion chromatography or, in the case of KIV 10 , lysine–Sepharose chromatography.

For size exclusion chromatography, fractions from the Ni 2+ –Sepharose column containing partially-purified KIV derivatives were applied to a Bio-Gel P-60 (Bio-Rad Laboratories; ~300 ml) column and resolved by gravity at 4°C. KIV-containing fractions (as determined by SDS–PAGE on 15% polyacrylamide gels followed by silver staining) were pooled and dialyzed against 20 mM Tris–HCl pH 7.9 for 1–2 days (minimum of four changes of buffer). The protein was then incubated in the presence of oxidized and reduced glutathione (1.25 mM each) for 2 days to ensure complete protein refolding. For some preparations, refolding was performed on the Bio-Gel P-60 eluate prior to its dialysis.

For purification of wild-type KIV 10 , Ni 2+ –Sepharose chromatography was followed by purification over a lysine–Sepharose column. Initially, a lysine–Sepharose CL-4B (Pharmacia) column (2.5 ml) was equilibrated with 10 column volumes of 20 mM Tris–HCl pH 7.9 containing 0.5 M NaCl. Fractions from the Ni 2+ –Sepharose column containing partially-purified KIV 10 were applied to the resin as described above, and the column was subsequently washed with 10 column volumes of 20 mM Tris–HCl pH 7.9 containing 0.5 M NaCl. Specifically-bound protein was eluted with 20 mM Tris–HCl pH 7.9 containing 0.5 M NaCl and 0.2 M of the lysine analogue {varepsilon}-aminocaproic acid ({varepsilon}-ACA). Fractions containing KIV 10 (as determined by SDS–PAGE on 15% polyacrylamide gels followed by silver staining) were pooled and dialyzed against 20 mM Tris–HCl pH 7.9 (minimum of four changes of buffer).

The integrity of the purified proteins was determined by SDS–PAGE on 15% gels followed by staining with silver or Coomassie blue. Protein concentrations for the KIV 2 , KIV 7 , KIV 9 {Delta}Cys and KIV 10 derivatives were each calculated by measurement of the absorbance at 280 nm (A 280 ) using estimated molar extinction coefficients as determined by the method of Fodor et al. (1989) for the respective proteins expressed in human embryonic kidney (293) cells (Sangrar et al., 1994; W.Sangrar, unpublished results ). The molar extinction coefficient estimated for the wild-type KIV 10 protein was used to determine the concentrations of the KIV 10 mutant derivatives. The molecular weights and extinction coefficients used for calculating protein concentrations were as follows: KIV 2 [MW = 17 528, {varepsilon} 0.1% (280 nm) = 1.59]; KIV 7 [MW = 18 052, {varepsilon} 0.1% (280 nm) = 2.12]; KIV 9 {Delta}Cys [MW = 17 893, {varepsilon} 0.1% (280 nm) = 2.12]; KIV 10 [MW = 17 821, {varepsilon} 0.1% (280 nm) = 1.27]; KIV 10 Trp 70 ->Arg [MW = 17 791, {varepsilon} 0.1% (280 nm) = 1.27]; KIV 10 Trp 70 ->Phe [MW = 17 782, {varepsilon} 0.1% (280 nm) = 1.27]; KIV 10 Arg 35 ->Lys [MW = 17 821, {varepsilon} 0.1% (280 nm) = 1.27].

Measurement of the lysine binding properties of KIV 10 derivatives

Lysine–Sepharose CL-4B (Pharmacia) columns (250 µl) were prepared and washed with 10 column volumes of 20 mM Tris–HCl pH 7.9. For each assay, protein (125 µg) was allowed to bind the column in a volume of 500 µl over a period of ~1 h with occasional resuspension of the resin. Unbound protein was collected in the flowthrough fraction and the column was subsequently washed with 20 mM Tris–HCl pH 7.9 until no protein was detected using a Bio-Rad protein assay (Bio-Rad Laboratories) on a 10 µl aliquot of each 250 µl fraction. The column was washed with 20 mM Tris–HCl pH 7.9 containing 0.5 M NaCl to elute weakly-bound protein until protein was no longer detectable in the column fractions. Specifically-bound protein was eluted by the addition of five column volumes of 20 mM Tris–HCl pH 7.9 containing 0.5 M NaCl and 0.2 M {varepsilon}-ACA. Elution fractions were analyzed for the presence of protein by subjecting aliquots of the fractions to SDS–PAGE on 15% polyacrylamide gels followed by silver staining. For lysine–Sepharose chromatography of the KIV 10 Trp 70 ->Phe and KIV 10 Arg 35 ->Lys species, Western blot analyses were also performed in order to quantitate the efficiency of binding of these derivatives to the column. Following SDS–PAGE, proteins were transferred onto an Immobilon-P nylon membrane (Millipore); the membrane was probed with an anti-apo(a) specific polyclonal antibody raised in rabbits (Rahman et al., 1998; 1:5000 dilution ). Apo(a)-immunoreactive material was detected using a secondary donkey anti-rabbit antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech; 1:5000 dilution); bands were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech) and quantified by densitometry. Densitometric analysis of the immunoblots was performed using a Hewlett Packard Scanjet 3c flatbed scanner and analyzed using Corel Photopaint (version 7.0, Corel Corp.) and Sigmagel (version 1.0, Jandel Scientific). The percentage of protein eluted under specific buffer conditions was determined by dividing the sum of the density of the respective fractions by the total amount of protein observed.

Measurement of the binding of apo(a) KIV domains to plasmin-modified fibrinogen

Binding assays were performed essentially as previously described (LoGrasso et al., 1994Go ; Sangrar et al., 1997Go ) with minor modifications. Purified human fibrinogen and human plasmin were generously provided by Dr Michael Nesheim (Department of Biochemistry, Queen's University). Microtiter wells were coated with 100 µl of fibrinogen [10 µg ml –1 in HEPES-buffered saline (HBS)] for 90 min at 37°C. Wells were washed four times with 150 µl of PBS containing 0.1% Tween-20 (PBST) after this and subsequent incubations. The fibrinogen-coated wells were treated with 100 µl of plasmin [30 ng ml –1 in HBS containing 0.1% Tween-20 (HBST)] for 30 min at 37°C. Wells were washed twice with 150 µl HBST containing 0.5 M NaCl and 0.2 M {varepsilon}-ACA prior to PBST washes. Residual plasmin was inactivated by incubation with 100 µl well –1 of D -Val-Phe-Lys-chloromethyl ketone (VFKck) dihydrochloride (Calbiochem; 1 µM in HBST) for 40 min at room temperature. Following PBST washes, non-specific binding sites were blocked overnight at 4°C by treatment with 150 µl PBS containing 2% BSA (Sigma). Immobilized plasmin-modified fibrinogen was incubated in the presence of increasing concentrations of apo(a) KIV in diluent buffer (HBST containing 1% BSA) for 2 h at room temperature. A 17-kringle-containing recombinant apo(a) [17K r-apo(a)] derivative containing eight copies of KIV 2 (Koschinsky et al., 1991Go ) was included as a positive control. Bound protein was detected by incubation with a rabbit anti-apo(a) polyclonal antibody (Rahman et al., 1998; 1:5000 dilution ) for 1 h at room temperature, followed by incubation for 1 h with a secondary donkey anti-rabbit antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, 1:5000 dilution). Binding of the secondary antibody was detected by addition of 100 µl/well development buffer containing 0.42 mg ml –1 o -phenylenediamine dihydrochloride (OPD, Sigma). Colour development was arrested by the addition of 50 µl/well 2 M H 2 SO 4 and the absorbance at 490 nm (less the background absorbance at 650 nm) was determined using a Titertek plate reader. Assays were performed in duplicate and resulting values averaged. Specific binding was determined by subtracting the signal obtained for wells not coated with fibrinogen. In an additional series of experiments, binding assays were performed as described above in the presence of 0.2 M of {varepsilon}-ACA, L -lysine, L -arginine or L -proline, or 50 mM L -phenylalanine.

Determination of the effect of apo(a) KIV domains on fibrinolysis

In vitro clot lysis assays were performed essentially as previously described (Sangrar et al., 1995Go ). Human fibrinogen (hFgn), plasminogen (hPgn), thrombin (IIa) and recombinant tPA (r-tPA) were generously provided by Dr Michael Nesheim (Department of Biochemistry, Queen's University). Clots were formed in a final volume of 200 µl in microtitre wells (Immulon-4; VWR Scientific) in HBS containing 0.01% Tween-80. To each well was added 180 µl of a solution containing hFgn (final concentration 1.03 mg ml –1 ), hPgn (final concentration 0.68 µM), CaCl 2 (final concentration 10 mM) and increasing concentrations of the respective KIV derivatives (final concentration 0–10 µM). Clot formation was effected by addition of 20 µl of a solution containing IIa (final concentration 6 nM) and r-tPA (final concentration 3 pM). The concentration of r-tPA was optimized to give an average clot lysis time of ~100 min. Lysis of the resultant fibrin clots was monitored by measurement of the absorbance (405 nm, 37°C) at 2 min intervals in a Titertek Twin reader using the SoftMax program. Lysis time was defined as the transition midpoint ( t m ), the point on the lysis curve where the absorbance is halfway between the maximum and minimum excursions with the percentage increase in t m defined as {Delta} t m . Lysis was also measured in the absence of r-tPA in the presence and absence of each KIV derivative at the respective maximum concentration; no lysis was observed indicating that, under these conditions, fibrinolysis is dependent upon exogenous tPA (data not shown). For some experiments, a slightly modified procedure was used for clot formation. Briefly, microaliquots (2 µl) of CaCl 2 , IIa and r-tPA (or buffer for the negative controls) were added to microtiter wells. A solution (194 µl) of hFgn and hPgn containing increasing concentrations of the respective apo(a) KIV derivatives was then added to the wells; clot formation and lysis were monitored as described above. For all assays, data were normalized using the following equation:

where A b is the basal absorbance (measured after complete clot lysis) and A m is the maximal absorbance (corresponding to the fully formed fibrin clot).

Molecular modeling of the KIV 10 Trp 70 ->Phe and KIV 10 Arg 35 ->Lys mutant derivatives

Modeling studies of the binding sites of the KIV 10 Trp 70 ->Phe and KIV 10 Arg 35 ->Lys mutant derivatives were based upon the previously published structure of the KIV 10{varepsilon}-ACA complex (Thr 64 variant ) (Mochalkin et al., 1999Go ). All modeling analyses were carried out using Sybyl, Version 5.3 (Tripos Inc., St Louis). For each derivative, the relevant amino acid was modified from the wild-type sequence and water molecules were removed from the crystal structure. Energy minimization was then performed using the energy gradient determination method employed in Sybyl. Diagrams were generated using MOLSCRIPT ( Kraulis, 1991Go ).


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Expression and purification of recombinant apo(a) KIV domains and mutant derivatives

We have previously described the use of the pET expression system for the production of single apo(a) kringle IV domains in E.coli (Rahman et al., 1998Go ). In this system, recombinant proteins are fused with 22 amino acids at the N-terminus which include a 10-residue histidine tag to facilitate purification by metal chelation. In order to characterize the functional contribution of single KIV domains to the effect of apo(a) on fibrinolysis and to the binding of apo(a) to plasmin-modified fibrinogen surfaces, we used this system to express apo(a) KIV 2 , KIV 7 , KIV 9 {Delta}Cys (in which the free cysteine at position 67 has been mutated to tyrosine) and KIV 10 in E.coli BL21 (DE3) cells. The apo(a) kringles were solubilized from bacterial inclusion bodies, refolded and purified to homogeneity using Ni 2+ –Sepharose followed by either gel filtration chromatography or lysine–Sepharose affinity chromatography. The purified kringle domains exhibited a characteristic mobility shift on SDS–PAGE in the presence of 10 mM DTT which is thought to correspond to the unfolding of the kringle motif under reducing conditions (Figure 1A Go ) (Sangrar et al., 1994Go ).



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Fig. 1. Expression of apo(a) KIV domains in E.coli . In all cases, recombinant proteins were expressed and purified from the insoluble fraction of transformed BL21 (DE3) cells as described in Materials and methods. Purified proteins (A, 5 µg; B–D, 2 µg) were resolved by SDS–PAGE on 15% polyacrylamide gels under non-reducing (NR) or reducing (R; containing 10 mM DTT) conditions and were visualized by staining with Coomassie Blue ( A ) or silver (B–D). The positions of molecular mass markers (Bio-Rad) are indicated to the left of each gel. ( A ) KIV 2 , KIV 7 , KIV 9 {Delta}Cys, KIV 10 . ( B ) KIV 10 Trp 70 ->Arg. ( C ) KIV 10 Trp 70 ->Phe. ( D ) KIV 10 Arg 35 ->Lys.

 
It has been speculated that the pathogenic role of Lp(a) is attributable, at least in part, to the lysine-binding ability of apo(a) which is mediated primarily by the LBS present in KIV 10 . In order to confirm the functional role of this LBS, we have engineered and expressed a series of mutant derivatives of KIV 10 in E.coli (Figure 1B–D Go ). The substitution of the tryptophan residue at position 70 (Trp 70 ) with arginine, as observed in Rhesus monkey apo(a) (Tomlinson et al., 1989Go ; Scanu et al., 1993Go ) as well as in a small percentage of the human population ( Scanu and Edelstein, 1994Go ; Scanu et al., 1994Go ) has been associated with plasma Lp(a) that is defective in lysine binding. This has been postulated to occur due to the key structural role of this tryptophan residue in the hydrophobic trough of the LBS (reviewed in Scanu and Edelstein, 1995). Thus, in our study a KIV 10 derivative was engineered to replace this critical Trp 70 residue with Arg (KIV 10 Trp 70 ->Arg ). A KIV 10 Trp 70 ->Phe mutant was also engineered to assess the effect of a more conservative substitution of Trp 70 . The KIV 10 Arg 35 ->Lys derivative contains a substitution of the Arg residue at position 35 to a Lys; the LBS of plasminogen KIV also contains a lysine residue at this position (Wu et al., 1991Go ).

Analysis of binding of KIV 10 derivatives to lysine–Sepharose

In order to determine the lysine-binding properties of the KIV 10 mutant derivatives, we assessed their ability to bind to lysine–Sepharose columns. Purified recombinant proteins were incubated with lysine–Sepharose resin for ~1 h, after which unbound protein was removed by washing with 20 mM Tris–HCl pH 7.9. The resin was then washed in the presence of 0.5 M NaCl to elute weakly-bound protein. Specifically-bound protein was eluted by the addition of 0.2 M {varepsilon}-ACA. Protein in the respective fractions was visualized by SDS–PAGE followed by silver staining. Consistent with previous analyses in our laboratory utilizing KIV 10 expressed from 293 cells (Sangrar et al., 1994Go ), the wild-type KIV 10 protein binds completely to lysine–Sepharose and can only be removed from the column by the addition of {varepsilon}-ACA (Figure 2A Go ) . Substitution of Trp 70 with Arg abolishes the ability of the corresponding mutant KIV 10 species to bind to lysine–Sepharose; all of the KIV 10 Trp 70 ->Arg protein was present in the unbound fraction (Figure 2B Go ). Substitution of the Trp at position 70 of KIV 10 with a Phe appears to reduce the specific binding of the corresponding species to lysine–Sepharose. Quantitation of the fraction bound to the column was performed by Western blotting followed by densitometric analysis of the immunoreactive bands, and indicated that ~50% of the KIV 10 Trp 70 ->Phe derivative was present in the unbound fraction whereas the remaining ~50% was removed from the column by the addition of 0.5 M NaCl (Figure 2C Go ). Replacement of the Arg at position 35 in KIV 10 with a Lys had a relatively minor effect on the ability of the corresponding derivative to bind to lysine–Sepharose. Indeed, the majority of the KIV 10 Arg 35 ->Lys derivative (~70%) was present in the {varepsilon}-ACA eluate with a small proportion (~30%) present in the 0.5 M NaCl wash fractions (Figure 2D Go ).



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Fig. 2. The binding of KIV 10 derivatives to lysine–Sepharose. Purified KIV 10 derivatives were each incubated with lysine–Sepharose for ~1 h in 20 mM Tris–HCl pH 7.9 and the flowthrough (F) collected. Unbound protein was washed from the resin with 20 mM Tris–HCl pH 7.9 (Wash). Weakly-bound protein was removed by the addition of 20 mM Tris–HCl pH 7.9 containing 0.5 M NaCl. Specifically-bound protein was eluted with 20 mM Tris–HCl pH 7.9 containing 0.5 M NaCl and 0.2 M {varepsilon}-ACA. Column fractions were analyzed by SDS–PAGE on 15% polyacrylamide gels followed by silver staining (A and B) or Western blotting using an anti-apo(a) polyclonal antibody (C and D). ( A ) KIV 10 . ( B ) KIV 10 Trp 70 ->Arg. ( C ) KIV 10 Trp 70 ->Phe. ( D ) KIV 10 Arg 35 ->Lys.

 
Determination of the binding of apo(a) KIV domains to plasmin-modified fibrinogen

Both apo(a) as well as the single KIV 10 domain have been shown to bind plasmin-modified fibrinogen surfaces (LoGrasso et al., 1994Go ; Sangrar and Koschinsky, 2000Go ). However, while the KIV 10 domain may provide a major component of the binding of apo(a) to fibrinogen (LoGrasso et al., 1994Go ; Boonmark et al., 1997Go ), it is postulated that a site outside this domain may also contribute to the interaction (Klezovitch et al., 1996Go ; Scanu et al., 1997Go ; Xue et al., 1999Go ). Thus, the ability of individual single kringle domains to interact with plasmin-modified fibrinogen was determined. Fibrinogen was immobilized in microtitre wells, treated with plasmin, and incubated with increasing concentrations of the respective KIV derivatives. Bound protein was detected by ELISA using a polyclonal antibody specific for apo(a). The ability to bind fibrinogen was assessed in parallel by incubation of recombinant KIV derivatives with immobilized fibrinogen which had not been treated with plasmin. Both a 17-kringle form of recombinant apo(a) [17K r-apo(a)] as well as KIV 10 showed saturable binding to fibrinogen. Plasmin treatment of the fibrinogen increased the binding of both KIV 10 and 17K r-apo(a) (Figure 3A Go ) . No binding of either KIV 2 or KIV 9 {Delta}Cys to intact or plasmin-treated fibrinogen was observed (Figure 3B Go ). KIV 7 bound very weakly, if at all, to fibrinogen; plasmin treatment of the fibrinogen resulted in significant and saturable binding of KIV 7 to the surface (Figure 3B Go ). However, the binding saturation of KIV 7 occurred at a much higher protein concentration (~2 µM) compared with that of KIV 10 (~100 nM), suggesting that the former kringle binds much less avidly to plasmin-modified fibrinogen.





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Fig. 3. The binding of recombinant apo(a) KIV derivatives to plasmin-modified fibrinogen surfaces. Binding of purified KIV derivatives to fibrinogen (open symbols) and plasmin-modified fibrinogen (closed symbols) was determined by ELISA using an anti-apo(a) polyclonal antibody raised in rabbits. Bound primary antibody was detected using a donkey anti-rabbit secondary antibody conjugated to horseradish peroxidase. Following a colorimetric assay for horseradish peroxidase, the absorbance at 490 nm was determined for each well and corrected for the absorbance at 650 nm. The assays were performed in duplicate and the resulting values were averaged. Specific binding was determined by subtracting the signal obtained for wells not coated with fibrinogen. ( A ) Binding of 17K r-apo(a) (diamonds) and KIV 10 (squares) to fibrinogen and plasmin-modified fibrinogen; ( B ) binding of KIV 2 (diamonds), KIV 7 (squares) and KIV 9 {Delta}Cys (triangles) to fibrinogen and plasmin-modified fibrinogen surfaces; ( C ) binding of KIV 10 derivatives KIV 10 Trp 70 ->Arg (diamonds), KIV 10 Trp 70 ->Phe (squares) and KIV 10 Arg 35 ->Lys (triangles) to fibrinogen and plasmin-modified fibrinogen.

 
To characterize further the interaction of KIV 10 with fibrinogen, the ability of the various mutant derivatives of this kringle to bind to fibrinogen and plasmin-modified fibrinogen was also assayed (Figure 3C Go ). Binding to both intact and plasmin-modified fibrinogen was completely abolished by the substitution of Trp 70 with an Arg (KIV 10 Trp 70 ->Arg ). Both KIV 10 Trp 70 ->Phe and KIV 10 Arg 35 ->Lys exhibited a reduced ability to bind to plasmin-modified fibrinogen, reaching saturation at concentrations of ~2 µM rather than the ~100 nM observed with the wild type. Similar to the wild-type KIV 10 , both mutant derivatives also bound intact fibrinogen to a lesser extent than plasmin-modified fibrinogen.

Effect of amino acids on the binding of KIV domains to plasmin-modified fibrinogen

In order to characterize further the binding of the KIV derivatives to immobilized fibrinogen and plasmin-modified fibrinogen, binding assays were performed in the presence of 0.2 M {varepsilon}-ACA, L -arginine and L -proline, or 50 mM L -phenylalanine; the use of higher concentrations of phenylalanine was limited by its solubility. Binding of both KIV 7 and KIV 10 to plasmin-modified fibrinogen was inhibited by {varepsilon}-ACA and L -arginine, while L -phenylalanine had no significant effect at the concentration used (Figure 4A and B Go ) . Interestingly, the binding of the two kringles to plasmin-modified fibrinogen differed with respect to sensitivity to proline addition. Binding of KIV 7 was ~90% inhibited by the addition of L -proline, yet this amino acid had no appreciable effect on the binding of KIV 10 to plasmin-modified fibrinogen. Thus, KIV 7 contains a lysine- and proline-sensitive binding site which may correspond to the fibrinogen binding site previously reported by Klezovitch et al. (1996). The apparent increase in the binding of KIV 10 in the presence of L -proline is due to a decrease in non-specific binding; the total apparent binding remained the same (data not shown). The effects of amino acids on the binding of KIV 10 Trp 70 ->Phe (Figure 4C Go ) and KIV 10 Arg 35 ->Lys (Figure 4D Go ) to plasmin-modified fibrinogen were similar to their effects on the binding of wild-type KIV 10 : {varepsilon}-ACA and L -arginine completely abolished the binding while L -proline had little or no effect. In general, the effects on the binding of KIV 10 mutants to plasmin-modified fibrinogen resulting from the addition of the respective amino acids were similar to those observed with the unmodified fibrinogen surface (data not shown).






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Fig. 4. The effect of addition of amino acids on the binding of apo(a) KIV derivatives to plasmin-modified fibrinogen surfaces. Binding assays were performed as described in the legend to Figure 3 Go for KIV 7 ( A ), KIV 10 ( B ), KIV 10 Trp 70 ->Phe ( C ) or KIV 10 Arg 35 ->Lys ( D ), in the absence (diamonds) and presence of 0.2 M {varepsilon}-ACA (squares), L -arginine (circles) or L -proline (asterisks), or 50 mM L -phenylalanine (triangles).

 
Determination of the effect of apo(a) KIV domains on fibrinolysis

Previous studies in our laboratory demonstrated a dose-dependent antifibrinolytic effect of 17K r-apo(a) in fibrin clots formed using purified components (Sangrar et al., 1995Go ). In the present study, this system was used to examine the effect of single KIV domains on fibrinolysis in vitro . Fibrin clots were formed in the wells of microtitre plates and tPA-mediated clot lysis was monitored in the presence of increasing concentrations of the respective KIV derivatives. Clot formation is marked by an initial rapid increase in turbidity as measured by the absorbance at 405 nm. Subsequent clot lysis is reflected by a rapid decrease in the turbidity signal to a baseline level. Inclusion of KIV 10 in the clot lysis assay resulted in a dose-dependent antifibrinolytic effect (Figure 5A Go ) , with a calculated {Delta} t m of ~43% observed in the presence of 10 µM of the kringle. Interestingly, KIV 10 Trp 70 ->Phe and KIV 10 Arg 35 ->Lys, as well as KIV 7 , which all bound relatively weakly to plasmin-modified fibrinogen (Figures 3B and C Go ), did not affect clot lysis time (Figure 5B–D Go ). Similarly, KIV 2 , KIV 9 {Delta}Cys and KIV 10 Trp 70 ->Arg had no significant effect on clot lysis time (data not shown).






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Fig. 5. The effect of apo(a) KIV domains on fibrinolysis in vitro . Fibrin clots were formed in microtitre wells using purified components as described in Materials and methods and the lysis of the clots was monitored by measurement of absorbance at 405 nm using a Titertek plate reader. Clots were formed in a final volume of 200 µl and contained fibrinogen (1.03 mg ml –1 ), plasminogen (0.68 µM), CaCl 2 (10 mM), thrombin (6 nM) and r-tPA (3 pM) in the absence and presence of increasing concentrations of KIV. ( A ) Clot lysis in the absence (diamonds) and presence of 0.5 µM (squares), 1 µM (triangles), 2 µM x 5 µM (asterisk) and 10 µM (circles) of KIV 10 . Inset: {Delta} t m values [defined as the percentage increase in the transition midpoint ( t m )] plotted as a function of KIV 10 concentration in the clots. ( BD ) Clot lysis in the absence (open symbols) and presence of 10 µM (closed symbols) of KIV 10 Trp 70 ->Phe (B), KIV 10 Arg 35 ->Lys (C) or KIV 7 (D).

 
Molecular modeling of the KIV 10 Trp 70 ->Phe and KIV 10 Arg 35 ->Lys mutant derivatives

Molecular models of the structures of the KIV 10 Trp 70 ->Phe and KIV 10 Arg 35 ->Lys mutant derivatives were constructed based on the known crystal structure of the KIV 10{varepsilon}-ACA complex (Mochalkin et al., 1999Go ) (Figure 6A Go ) . Due to the unavailability of the crystal structure for the Met 64 variant of KIV 10 complexed with {varepsilon}-ACA, the Thr 64 variant was used both to generate the molecular models of the respective mutant derivatives as well as to compare the effects of the single mutations on the structures of the respective LBS as previous studies have shown that the two variants are highly isostructural (Mochalkin et al., 1999Go ).



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Fig. 6. Molecular modeling of KIV 10 Trp 70 ->Phe and KIV 10 Arg 35 ->Lys mutant derivatives. ( A ) Ribbon diagram of apo(a) KIV 10 complexed with {varepsilon}-ACA. The side chains of all residues implicated in ligand interactions are shown (beige). The KIV 10 backbone is shown in green and {varepsilon}-ACA is shown in yellow. ( B ) Superimposition of the models of KIV 10{varepsilon}-ACA and the KIV 10 Trp 70 ->Phe derivative in which Trp 70 (beige) has been replaced with Phe (pink). The color scheme for KIV 10{varepsilon}-ACA is as described for (A). The KIV 10 Trp 70 ->Phe backbone is shown in orange. ( C ) Superimposition of the models of KIV 10{varepsilon}-ACA and the KIV 10 Arg 35 ->Lys derivative. Color scheme for KIV 10{varepsilon}-ACA is as described for (A). The KIV 10 Arg 35 ->Lys backbone is shown in orange. Replacement of Arg 35 (beige) with Lys (pink) in the mutant derivative results in movement of the side chain of Asp 54 (purple) relative to its corresponding position in the wild-type protein (beige). For clarity, in both (B) and (C), only residues which differ in identity and/or position between the wild-type and mutant derivatives are shown.

 
Comparison of the crystal structure of KIV 10 with that of the model of the KIV 10 Trp 70 ->Phe mutant derivative revealed that, as expected, the two residues at position 70 are superimposable (Figure 6B Go ). In addition, no significant changes were predicted in the structure of the LBS relative to that of the wild-type protein. The smaller structure of Phe compared with Trp, however, would readily result in a decrease in the hydrophobic interactions involved in stabilizing the aliphatic backbone of the ligand.

The side chain of the Lys 35 residue in the model of the KIV 10 Arg 35 ->Lys mutant derivative (Figure 6C Go ) is superimposable with the guanidinium group of the corresponding Arg residue in the KIV 10 crystal structure. However, this substitution results in the movement of Asp 54 away from the amino group of the ligand. To ensure that this movement was not a result of the removal of water molecules, as a control, energy minimization of the wild-type KIV 10 structure was performed following removal of water molecules from the crystal structure. In the wild-type protein, steric hindrance due to the bulkier side chain of Arg 35 may constrain the movement of Asp 54 so as to `force' its interaction with the ligand; the distance between the side chain of Asp 54 in the mutant derivative and the guanidinium group of Arg 35 in the wild-type protein was found to be 1.7 Å, which is too close to allow salt bridge formation between these two residues. The functional {varepsilon}-amino group of Lys 35 was also found to be situated further away from the carboxyl end of the ligand relative to the guanidinium group of Arg 35 in the wild-type protein, which may also decrease ligand stabilization in the mutant.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One mechanism by which Lp(a) is thought to mediate its pathogenic effects involves the ability of the apo(a) component of the lipoprotein particle to inhibit plasminogen activation on fibrin surfaces either by complexing with plasminogen in solution, thereby reducing the amount of plasminogen bound to fibrin (Sangrar et al., 1997Go ), or by direct competition with plasminogen for lysine-mediated binding sites on fibrin (Rouy et al., 1992Go ; Hervio et al., 1995Go ). Based on the latter scenario, several studies have been performed to identify the domain(s) of apo(a) which interact with fibrin(ogen). The ability of lysine-binding defective Lp(a) species from either human or Rhesus monkeys [containing a critical Trp 70 ->Arg substitution in the LBS of apo(a) KIV 10 ] to bind plasmin-modified fibrinogen led investigators to conclude that this process involves a lysine- and proline-sensitive domain that is outside the LBS of apo(a) KIV 10 ( Klezovitch et al., 1996Go ). Subsequent studies using proteolytically-derived apo(a) fragments localized this domain to a weak LBS within the KIV 5 –KIV 8 region, and suggested an important role for KIV 8 in apo(a)-fibrinogen interactions ( Scanu et al., 1997Go ). Conversely, in studies using purified recombinant apo(a) KIV 10 ( LoGrasso et al., 1994Go ), it was demonstrated that this domain can interact directly with plasmin-modified fibrinogen surfaces with an observed EC 50 of 14 ± 1.2 µM; the corresponding EC 50 for the interaction of Lp(a) with these surfaces was found to be 1.0 ± 0.3 nM. These findings suggest that the lysine-dependent interaction of apo(a) with fibrin(ogen) is primarily mediated by KIV 10 , which contains the strongest LBS in apo(a), with the contribution of one additional weak LBS in apo(a) being both reasonable and adequate to account for the overall interaction of Lp(a) (LoGrasso et al., 1994Go ). This is consistent with analyses which demonstrated that an apo(a) derivative lacking a functional LBS in KIV 10 (due to substitution of both Asp 54 and Asp 56 with Ala ) exhibits a reduction of binding to plasmin-modified fibrin of ~60% relative to wild-type apo(a) (Boonmark et al., 1997Go ).

In the present study we have examined the interaction of individual KIV domains of apo(a) with plasmin-modified fibrinogen in order to ascertain their contribution to the overall interaction. Moreover, we examined the ability of these domains to mediate the antifibrinolytic effect of apo(a) which has been reported both in vitro (Sangrar et al., 1995Go ) and in vivo (Palabrica et al., 1995Go ; Biemond et al., 1997Go ). The LBS of plasminogen KI and KIV are largely responsible for binding fibrin, and bind to C-terminal lysine residues which are exposed as fibrin becomes degraded by plasmin. While apo(a) KIV 10 is known to contain the strongest LBS in apo(a), we have demonstrated that KIV 7 contains a weaker LBS, while KIV 2 has no affinity for lysine (M.N.Rahman and M.L.Koschinsky, in preparation). Further, we have shown that KIV 9 {Delta}Cys can interact weakly with lysine–Sepharose (M.N.Rahman and M.L.Koschinsky, unpublished data). In the present study, we have demonstrated that both KIV 7 and KIV 10 can interact with plasmin-modified fibrinogen while KIV 2 and KIV 9 {Delta}Cys showed little if any binding. The degree of interaction of KIV 7 and KIV 10 with plasmin-modified fibrinogen appeared to be correlated with the strength of the LBS: the binding of KIV 10 to plasmin-modified fibrinogen reached saturation at a concentration ~10-fold lower than KIV 7 (Figure 3A and B Go ) and KIV 10 binds {varepsilon}-ACA with an ~10-fold higher affinity ( K D ~33 µM ) than KIV 7 ( K D ~230 µM ) (M.N.Rahman and M.L.Koschinsky, in preparation).

To characterize further the correlation of lysine-binding affinity of the KIV derivatives with the ability to interact with plasmin-modified fibrinogen, various mutants of KIV 10 were examined. The LBS of KIV 10 (Figure 6A Go ) contains a hydrophobic trough located at the kringle surface which is lined by Trp 60 , Phe 62 and Trp 70 , and which acts to stabilize the aliphatic backbone of T-amino acids such as lysine. Anionic (Asp 54 /Asp 56 ) and cationic (Arg 35 /Arg 69 ) charge pairs at either end of the trough interact with the respective charged groups of zwitterionic ligands (reviewed in Scanu and Edelstein, 1995). The Trp 70 residue appears to play a significant role in ligand stabilization; its indole group is partially exposed to the solvent and, along with the two charged clusters of the LBS, serves as a recognition marker for lysine (reviewed in Scanu and Edelstein, 1995). Substitution of Trp 70 with Arg (KIV 10 Trp 70 ->Arg ), as observed in Rhesus monkey apo(a) (Tomlinson et al., 1989Go ) as well as a small percentage of human apo(a) ( Scanu et al., 1994Go ), abolished the binding of KIV 10 to lysine–Sepharose (Figure 2B Go ). This was consistent with studies which have demonstrated the inability of both Rhesus Lp(a) and Lp(a) isolated from individuals containing this mutation, as well as KIV 10 containing the Trp 70 ->Arg substitution, to bind lysine–Sepharose (Scanu et al., 1993Go , 1994Go ; Klezovitch and Scanu, 1996Go ). Crystallographic studies have shown that the Arg 70 side chain extends along the ligand binding groove to mimic ligand binding (Mochalkin et al., 1999Go ). Thus, loss of lysine binding due to the Trp 70 ->Arg substitution is due to steric blockage of the LBS. The more conservative substitution of Trp 70 to a Phe residue, which should maintain the hydrophobic nature of the trough, led to a decrease in specific binding to the lysine–Sepharose resin (Figure 2C Go ). Molecular modeling of the KIV 10 Trp 70 ->Phe mutant derivative (Figure 6B Go ) revealed that this substitution does not alter the structure of the binding site significantly. Diminishment of the lysine affinity of the LBS may result from a significant decrease in the stabilizing hydrophobic interactions with the aliphatic backbone of the ligand due to the smaller hydrophobic surface area of Phe relative to Trp. Interestingly, the LBS of plasminogen KI, which exhibits a lysine affinity two orders of magnitude greater than that of plasminogen KIV, contains a tyrosine residue at this position. Replacement of this tyrosyl residue in plasminogen KI with phenylalanine does not significantly influence lysine binding while replacement with tryptophan results in a >10-fold increase in its lysine binding affinity (Hoover et al., 1993Go ). The ability of these KIV 10 Trp 70 mutant derivatives to bind plasmin-modified fibrinogen correlated with their ability to bind lysine–Sepharose. Comparison of the ability to bind plasmin-modified fibrinogen revealed that KIV 10 Trp 70 ->Phe required much higher levels of protein to reach binding saturation in comparison with the wild-type KIV 10 (~2 µM versus ~100 nM) while binding was negligible for KIV 10 Trp 70 ->Arg (Figure 3C Go ).

The LBS of KIV 10 (Figure 6A Go ) contains all the residues implicated in lysine coordination in plasminogen KIV with the exception of Arg 35 , which corresponds to a lysine residue in plasminogen KIV. Since plasminogen KI also contains an arginine at this position, Arg 35 in apo(a) KIV 10 has been suggested to account for the apparent KI-like binding specificity exhibited by this kringle (Hoover-Plow et al., 1993Go ). However, mutagenesis studies on both plasminogen KI and KIV have suggested that this residue plays a non-essential role in ligand binding in solution. Indeed, while an important role for the {varepsilon}-amino group of Lys 35 was suggested from the crystal structure of plasminogen KIV complexed with {varepsilon}-ACA (Wu et al., 1991Go ), the crystal structure of plasminogen KI suggested that the equivalent Arg 34 residue is non-essential as its guanidinium group is disordered and situated in the solvent region outside the LBS ( Wu et al., 1994Go ). Surprisingly, our results demonstrated that substitution of the Arg 35 in KIV 10 with a lysine, as found in plasminogen KIV, reduces its binding to plasmin-modified fibrinogen approximately to the same extent as KIV 10 Trp 70 ->Phe, requiring protein concentrations of ~2 µM to achieve binding saturation (Figure 3C Go ). Moreover, ~30% of the protein could be removed from lysine–Sepharose in the presence of 0.5 M NaCl (Figure 2D Go ), thereby implying a reduced affinity of the LBS in this derivative for lysine–Sepharose. Comparison of the crystal structures of plasminogen KI (Wu et al., 1994Go ) and apo(a) KIV 10 (Mikol et al., 1996Go ; Mochalkin et al., 1999Go ) reveals that the respective guanidinium groups corresponding to Arg 35 (i.e. Arg 34 in KI ) are superimposable (data not shown), which may suggest a similar non-essential role for this residue in KIV 10 . However, crystallographic studies indicate that upon binding of {varepsilon}-ACA to KIV 10 , the Arg 35 group undergoes a conformational change, resulting in movement of the guanidinium group towards the carboxylate group of the ligand to assist Arg 69 in stabilizing its anionic end (Mochalkin et al., 1999Go ). Indeed, of the residues situated within 5 Å of the LBS, only Arg 35 undergoes a conformational change upon ligand binding which is accompanied by a shift in the tyrosyl ring of Tyr 40 to fill the resultant vacancy (Mochalkin et al., 1999Go ). Replacement of the arginine with lysine may serve to decrease the number of interactions available with the ligand, thereby reducing its stabilization. However, molecular modeling of the KIV 10 Arg 35 ->Lys mutant derivative (Figure 6C Go ) revealed that this substitution results in the movement of Asp 54 , one of the components of the anionic center of the LBS, away from the amino group of the ligand. In the wild-type protein, steric hindrance due to the bulkier side chain of Arg 35 may constrain the movement of Asp 54 so as to `force' its interaction with the ligand whereas, in the mutant derivative, the presence of lysine at this position may allow Asp 54 more rotational flexibility. The {varepsilon}-amino group of Lys 35 was also found to be situated further away from the carboxyl end of the ligand relative to the guanidinium group of Arg 35 in the wild-type protein, which may also act to decrease ligand stabilization in the mutant derivative. Moreover, since the {varepsilon}-amino group of Lys 35 represents its sole functional group (cf. arginine), if it is involved in interactions with Asp 54 , Lys 35 may not be available to form stabilizing interactions with the ligand carboxylate group. It should be noted, however, that equilibrium ligand binding analyses ofKIV 10 Arg 35 ->Lys using intrinsic fluorescence indicate K D values for {varepsilon}-ACA (~21 µM) and L-lysine (~230 µM) (M.N.Rahman and M.L.Koschinsky, unpublished results) which are very similar to those of the wild-type protein (~33 and ~170 µM, respectively) (M.N.Rahman and M.L.Koschinsky, in preparation). This discrepancy may reflect differences in the binding of conformationally constrained ligands (such as lysine in lysine–Sepharose or plasmin-modified fibrinogen) versus conformationally free ligands (i.e. the zwitterionic species in solution).

Of the KIV domains examined, only wild-type apo(a) KIV 10 exhibited any significant effect on fibrinolysis (Figure 5 Go ). It is tempting to speculate that this inhibition reflects binding of KIV 10 to fibrin and thus inhibition of plasminogen binding. Indeed, neither KIV 7 nor the KIV 10 mutant derivatives, all of which exhibited reduced binding to fibrin, affected fibrinolysis. Recent studies have demonstrated that KIV 10 is capable of competing with both radiolabeled Lp(a) and plasminogen for binding to plasmin-modified fibrinogen (IC 50 values of ~17 and 14 µM, respectively ). However, both intact Lp(a) and plasminogen were more effective inhibitors for both interactions (IC 50 values of 7 and 370 nM for inhibition of plasminogen binding, respectively ) (Xue et al., 1999Go ). Similarly, the concentrations of KIV 10 required to inhibit fibrinolysis in the current study ({Delta} t m of 43% at 10 µM KIV 10 ) were substantially higher than the concentration of 17K r-apo(a) required to inhibit fibrinolysis in the study of Sangrar et al. (1995) [{Delta} t m of 64% at 0.27 µM 17K r-apo(a)]. These data imply that domains in apo(a) in addition to KIV 10 , such as the weak LBS in KIV 5–8 that mediate fibrin binding or the protease domain that mediates plasminogen binding, are required for the full antifibrinolytic effect of apo(a).

In addition to the potential mediation of the antifibrinolytic mechanism of apo(a), apo(a)–fibrin(ogen) interactions may contribute to the pathogenicity of Lp(a) by contributing to its retention in developing atherosclerotic plaques. Indeed, fibrinogen deficiency in apo(a) transgenic mice has been associated with a decrease in the vascular accumulation of apo(a) by 78% and average fatty streak lesion development by 81%. Moreover, fibrin(ogen) deposition was found to be virtually colocalized with sites of apo(a) deposition and fatty streak lesions in wild-type transgenic apo(a) mice ( Lou et al., 1998Go ). This trapping is likely to be enhanced at sites of injury due to the increased prevalence of residual thrombi. Indeed Lp(a) accumulation has been demonstrated in injured arterial tissue in both a cynomolgus monkey model ( Ryan et al., 1997Go ) and human tissues (Yano et al., 1997Go ), especially at fibrinogen-rich sites of thrombus formation and the `fibrous cap' formed in the second stage of inflammation. Determination of the role of the weak and strong LBS in apo(a) in mediating these interactions requires further analysis.

In summary, we have attempted to reconcile conflicting studies regarding the domains in apo(a) which mediate its interaction with fibrin(ogen) by examining recombinant KIV domains expressed in a bacterial system. We have confirmed that the strong LBS in KIV 10 is capable of mediating interactions with intact and plasmin-modified fibrinogen in a lysine- and arginine-sensitive manner, and demonstrated that it is capable of attenuating fibrinolysis in an in vitro clot lysis system. Examination of KIV 10 mutant derivatives revealed a correlation between lysine-binding capacity and the ability to interact with plasmin-modified fibrinogen. Moreover, destruction or diminishment of the lysine binding capacity of KIV 10 abolishes its antifibrinolytic effect. We have also shown that KIV 7 contains a lysine- and proline-sensitive site which is capable of binding to plasmin-modified fibrinogen, albeit to a lesser extent than KIV 10 , yet which does not affect fibrinolysis in vitro .


    Notes
 
1 To whom correspondence should be addressed. Email: mk11{at}post.queensu.ca Back


    Acknowledgments
 
Special mention and appreciation should be given to Lorraine F.May, Mark A.Hancock and Dr Jiazhi Xia for their contributions in generating the KIV 10 mutant expression plasmids utilized in this study. In addition, we wish to thank Dr Michael Nesheim, John Walker and Dr Michael Boffa for their generous donations of reagents and equipment utilized in this study. This work was funded by a grant from the Heart and Stroke Foundation of Ontario (#T-4408 to M.L.K.) and a grant from the Canadian Institutes of Health Research (Z.J.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received November 28, 2000; accepted March 5, 2001.





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