Apolipoprotein(a) Enhances Platelet Responses to the Thrombin Receptor–Activating Peptide SFLLRN

From the Department of Biochemistry, University of Toronto, Toronto (M.L.R., D.M.T., M.A.P.), and the Department of Biochemistry, Queen's University, Kingston (W.S., M.A.H., M.L.K.), Ontario, Canada; and Northwest Lipid Research Laboratories, University of Washington (S.M.M.), Seattle, Wash.

Correspondence to Dr M.L. Rand, Division of Haematology/Oncology, The Hospital for Sick Children, 555 University Ave, Toronto, Ontario, Canada M5G 1X8. E-mail margaret.rand{at}sickkids.on.ca

	Abstract

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Abstract—Elevated levels of lipoprotein(a) [Lp(a)] are correlated with an increased risk of atherosclerotic disease. We examined the effect of recombinant apolipoprotein(a) [r-apo(a)] and Lp(a) on responses of washed human platelets, prelabeled in the dense granules with [¹⁴C]serotonin and suspended in Tyrode's solution, to ADP and the thrombin receptor–activating peptide SFLLRN. No effect of the 17 kringle (K), 12K, or 6K r-apo(a) derivatives (at concentrations of 0.35 and 0.7 µmol/L) or Lp(a) (up to 0.1 µmol/L) on primary ADP-induced platelet aggregation was observed. In contrast, weak platelet responses stimulated by 7.5 µmol/L SFLLRN were significantly enhanced by the r-apo(a) derivatives; eg, 0.7 µmol/L 17K r-apo(a) increased aggregation from 15±4% to 58±6%, release of [¹⁴C]serotonin from 9±3% to 36±6%, and formation of thromboxane A₂, measured as its stable metabolite thromboxane B₂, from 7±1 to 29±5 ng/10⁹ platelets (n=3; P<0.04 to 0.015). Significant enhancement of aggregation and release of granule contents was observed at a concentration of 17K r-apo(a) as low as 0.175 µmol/L. Purified Lp(a) (0.25 to 0.1 µmol/L) also enhanced SFLLRN-induced aggregation and release in a dose-dependent manner. Although plasminogen (0.7 and 1.5 µmol/L) and low density lipoprotein (0.025 to 0.1 µmol/L) both exhibited potentiating effects on SFLLRN-mediated platelet aggregation, the magnitude of the responses was less than that observed with either the r-apo(a) derivatives or Lp(a). The enhanced responses of platelets via the protease-activated receptor-1 thrombin receptor in the presence of Lp(a) may contribute to the increased risk of thromboembolic complications of atherosclerosis associated with this lipoprotein.

Key Words: lipoprotein(a) • apolipoprotein(a) • platelet function • thrombin receptor–activating peptide • SFLLRN

	Introduction

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Elevated plasma levels of Lp(a) (ie, >20 to 30 mg/dL) have been identified as a significant risk factor for coronary artery disease, myocardial infarction, and infarcted artery patency.^{1 2} However, the mechanisms by which Lp(a) mediates its pathogenic effects are poorly understood and may involve prothrombotic or proatherogenic roles.^{1 2 3} Structurally, Lp(a) is very similar to LDL but is distinguished by the unique protein moiety apo(a), which is attached to apoB-100 by a single disulfide bridge.^{4 5} The primary sequence of apo(a) shares extensive homology with the serine protease zymogen plasminogen and contains multiply repeated copies of a sequence that closely resembles plasminogen kringle IV, followed by sequences exhibiting a high degree of similarity to the kringle V and protease domains of plasminogen.⁶ Ten distinct classes of kringle IV sequences in apo(a) are present in all individuals; the kringle IV type 2 motif (also known as the major repeat kringle) is present in variable numbers (<10 to >40) and constitutes the molecular basis of Lp(a) isoform size heterogeneity.^{7 8} Of the kringle IV sequences in apo(a), kringle IV type 10 most closely resembles that of plasminogen kringle IV. Like plasminogen kringle IV, apo(a) kringle IV type 10 also has lysine-binding properties⁹ and has been postulated to mediate the interaction of Lp(a) with lysine residues present in such biological substrates as fibrin.^{10 11 12}

The similarity between apo(a) and plasminogen has been interpreted as suggesting a potential prothrombotic/antifibrinolytic effect for Lp(a) that may underlie thromboembolic complications associated with elevated Lp(a) levels in vivo. Several studies have demonstrated that Lp(a) can compete with plasminogen for binding to fibrin surfaces^{10 11} and that both Lp(a)¹¹ and apo(a)¹³ inhibit plasminogen activation mediated by tissue plasminogen activator. It has also been demonstrated that Lp(a) binds to isolated platelets via a lysine-dependent interaction¹⁴ ; there are conflicting reports as to whether the glycoprotein (GP) IIb-IIIa complex is involved in Lp(a) binding to platelets.^{14 15} It was recently shown that Lp(a) inhibits collagen-induced platelet aggregation,^{16 17} perhaps by inhibition of platelet adhesion to collagen.¹⁶ Because LDL is generally considered to be proaggregatory with respect to platelets,^{18 19} these results suggest a role for the apo(a) component of Lp(a) in influencing platelet interactions.

In the current study, we examined the effect of recombinant apo(a) [r-apo(a)] derivatives differing in the number of kringle IV type 2 motifs on platelet responses (aggregation, secretion of granule contents, and formation of thromboxane A₂ [TXA₂]) mediated by ADP or the thrombin receptor–activating hexapeptide SFLLRN, 2 agonists that stimulate platelets via different biochemical mechanisms.²⁰ Our results clearly demonstrate that even though primary ADP-induced aggregation of platelets is not affected by the r-apo(a) species or isolated Lp(a), they significantly enhance aggregation, secretion of granule contents, and TX formation stimulated by SFLLRN. This enhancement is not affected by the number of kringle IV type 2 motifs in the r-apo(a) protein. These data illuminate a novel mechanism by which Lp(a) may contribute to thromboembolic complications associated with atherosclerosis.

	Methods

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Construction of Apo(a) Expression Plasmids
The 17 kringle (K), 12K, and 6K r-apo(a) derivatives used in this study are shown in Figure 1A; they differ in number of identically repeated kringle IV type 2 domains (8, 3, and 0, respectively). Details of the construction of the corresponding expression plasmids are described elsewhere.²¹ All constructs were assembled in the pRK5 expression vector, which contains the cytomegalovirus promoter and simian virus 40 termination sequences.²²

View larger version (36K): [in this window] [in a new window]   Figure 1. A, Composition of r-apo(a) species used. Top line, Organization of the 17K r-apo(a), which was derived from the published cDNA sequence as previously described.22 The organization of the 12K and 6K r-apo(a) derivatives is shown relative to the 17K species. In all cases, unfilled boxes designate kringle repeats of identical amino acid sequence (ie, kringle IV type 2); hatched boxes represent amino acids substitutions relative to the major kringle repeat. The 10 types of kringle IV sequences are indicated above the 17K derivative. Shaded boxes represent the kringle V sequence, and black bars correspond to the apo(a) protease-like domain (P). The position of the free cysteine in apo(a) kringle IV type 9 is shown with a bar. The 12K and 6K constructs contain a hybrid kringle (diagonal line), which represents a fusion of kringle IV type 1 with either kringle IV type 2 (for the 12K derivative) or kringle IV type 5 (for the 6K species). The small black rectangle at the left end of each construct represents the signal sequence. B, SDS-PAGE analysis of purified r-apo(a) derivatives. r-apo(a) was purified by lysine-Sepharose affinity chromatography from conditioned medium harvested from 293 cells stably expressing each derivative. The indicated recombinant proteins (5 to 10 µg) were analyzed by SDS-PAGE under nonreducing conditions using a 4% to 20% polyacrylamide gradient gel stained with Coomassie blue. Positions of molecular weight markers (Bio-Rad) are indicated to the left.

Generation of Stably Expressing Cell Lines
Human embryonic kidney (293) cells²³ (American Type Culture Collection, Rockville, Md) were cultured in 100-mm dishes in the presence of minimal essential medium (Gibco-BRL) supplemented with 5% FCS. Cell lines stably expressing the various r-apo(a) derivatives were generated by cotransfection of 293 cells with 10 µg of the respective expression plasmid and 1 µg of a plasmid encoding the neomycin resistance gene²⁴ per culture dish by calcium phosphate coprecipitation.²⁵ Transfectants were selected by culturing the cells in the presence of 800 µg/mL of the antibiotic G418 (Gibco-BRL) as previously described.²² Clones stably expressing the apo(a) derivatives were identified by ELISA.²¹

Protein Purification
Transfected 293 cells stably expressing the various r-apo(a) derivatives were cultured in roller bottles containing 250 mL of Optimem (Gibco-BRL) for 72 hours. Conditioned medium was harvested, clarified by brief centrifugation, and loaded onto a 50-mL lysine-Sepharose CL-4B (Pharmacia) column. The column was washed with PBS containing 0.5 mol/L NaCl, and protein was eluted with 0.2 mol/L -aminocaproic acid in this buffer. Protein-containing fractions were pooled, dialyzed at 4°C against HEPES-buffered saline (HBS; 20 mmol/L HEPES, pH 7.4, containing 150 mmol/L NaCl), precipitated with 50%(NH₄)₂SO₄, and pelleted by centrifugation at 12 000g for 20 minutes at 4°C. The resulting pellet was dissolved in HBS and dialyzed against the same buffer. The protein concentration was determined by measuring absorbance at 280 nm. Extinction coefficients for each r-apo(a) protein were determined by tyrosine difference spectra.²⁶

Glu-plasminogen was purified from fresh frozen plasma by adsorption to lysine-Sepharose CL-4B, followed by elution with -aminocaproic acid.²⁷ The dialyzed sample was precipitated with 70% (NH₄)₂SO₄, and the precipitate was recovered by centrifugation, dissolved in HBS, and dialyzed against HBS. Plasminogen ran as a single band of 92 kDa on a 4% to 20% SDS–polyacrylamide gel electrophoresis gel under reducing conditions. This preparation had no activity toward the plasmin-specific chromogenic substrate S-2251 (Kabi).

Lipoprotein Purification
To isolate Lp(a) from human plasma, blood samples were obtained from a fasting donor with high Lp(a) levels and an apo(a) isoform containing 19 kringle IV type 2 repeats, as determined by agarose gel electrophoresis and immunoblotting.^{28 29} Lp(a) was purified from the plasma by sequential density gradient ultracentrifugation, followed by gel filtration chromatography as previously described.³⁰ Purity of isolated Lp(a) was assessed by agarose gel electrophoresis, and the molar protein concentration was determined by a double monoclonal antibody–based ELISA insensitive to apo(a) size heterogeneity.³⁰ Additionally, the purified Lp(a) was determined to be free of contaminating plasminogen by immunoblotting (data not shown).

LDL within the 1.006<d<1.05 g/mL density range was isolated from human plasma by sequential flotation.³¹ In brief, plasma (containing 1 mmol/L PMSF, 1 mmol/L EDTA, and 0.02% NaN₃) was centrifuged at 436 000xg for 2 hours at 15°C. The d<1.006 g/mL fraction was removed, and the infranatant density was adjusted to <1.05 g/mL with NaBr and centrifuged for 2 more hours. At this time, the d<1.05 g/mL fraction was isolated and centrifuged at a density of 1.05 g/mL for another 2 hours. LDL isolated from this centrifugation step was found to be devoid of contaminating Lp(a), as determined by both immunoblotting and ELISA (data not shown). Lp(a) and LDL preparations were dialyzed extensively against HBS before they were used in platelet studies.

Preparation of Platelets
Suspensions of washed platelets were prepared from human donors who had not taken medication affecting platelet function for at least 2 weeks before the blood donation. (Informed consent was obtained from each subject, and experiments were approved by the University of Toronto Human Subjects Review Committee.) Blood (60 to 80 mL) was anticoagulated with the acid-citrate-dextrose solution of Aster and Jandl³² (88.4 mmol/L trisodium citrate, 71.4 mmol/L citric acid, and 111 mmol/L dextrose), using 1 part solution to 6 parts blood. The following preparation was performed at 37°C. Blood was centrifuged at 190g for 15 minutes to obtain the supernatant platelet-rich plasma. Platelet-rich plasma was centrifuged at 2500g for 15 minutes to obtain a platelet pellet. The pellet was gently resuspended in 10 mL of a washing solution based on Tyrode's solution (137 mmol/L NaCl, 2.7 mmol/L KCl, 11.9 mmol/L NaHCO₃, 0.42 mmol/L NaH₂PO₄, 1 mmol/L MgCl₂, 2 mmol/L CaCl₂, and 5.5 mmol/L glucose) containing heparin (50 U/mL), albumin (0.35%), and 10 µL apyrase,^{33 34} pH 7.35. To label the platelet dense granules with [¹⁴C]serotonin, 0.1 µCi/mL of 5-hydroxy-3-indolyl([1-¹⁴C]ethyl-2-amine)creatinine sulfate (55 mCi/mmol, Amersham) was incubated with this suspension for at least 15 minutes. The platelet pellet was recovered by centrifugation at 1200g for 10 minutes, and the platelets were resuspended in the second washing solution, which was the same as the first but without heparin. The platelets were recovered as before and finally resuspended in Tyrode-albumin solution containing 2 µL/mL apyrase, pH 7.35, at a platelet count of 0.35x10⁹ /mL. (The concentration of apyrase should be capable of converting 0.25 µmol/L ATP to AMP in 120 seconds at 37°C but should not have an appreciable effect on the extent of platelet aggregation induced by ADP in the presence of fibrinogen.) The platelet suspension was incubated for 30 minutes at 37°C before use. Imipramine (5 µmol/L) was added before initiation of platelet function studies (see below) to prevent reuptake of secreted [¹⁴ C]serotonin.

Aggregation of Platelets
Platelet aggregation was studied at 37°C in a Payton aggregation module (Ion Trace), which records light transmission through 0.5-mL samples of a platelet suspension stirred at 1100 rpm. Before addition of an agonist, baseline (zero aggregation) was set with the stirred platelet suspension, and 100% aggregation was set as light transmission through the suspending medium (without platelets). On addition of ADP (Sigma) or SFLLRN (synthesized by the Institute for Molecular Biotechnology, McMaster University), change in platelet shape was indicated by a small decrease in light transmission; this was followed by a large increase in light transmission as the platelets aggregated. In experiments with ADP, fibrinogen (100 µg/mL) was added to the samples 5 seconds before addition of ADP. The effects of r-apo(a), plasminogen, Lp(a), LDL, or diluent (HBS) were studied by adding them 15 seconds before the addition of ADP or SFLLRN. (Increasing the incubation time up to 30 minutes before addition of agonist did not affect the results.) The extent of platelet aggregation (expressed as a percentage) was indicated by the maximum increase in light transmission 3 minutes after the addition of ADP or 5 minutes after addition of SFLLRN.³⁵ All concentrations reported are final concentrations after all additions to the platelet suspensions.

Measurement of Secretion of Granule Contents and TX Formation
Three minutes after addition of ADP or 5 minutes after addition of SFLLRN, supernatant samples were prepared by centrifugation of the stimulated platelet suspensions for 1 minute at 12 000g in an Eppendorf centrifuge. These samples were used to determine the percentage of [¹⁴C]serotonin secreted from the prelabeled platelets and the formation of TXB₂, the stable metabolite of TXA₂, by radioimmunoassay (NEK-007, NEN Canada).

Statistical Analyses
Values are reported as mean±SEM with the number of experiments indicated. Paired t tests (with adjustment for multiple tests³⁶ when necessary) were used to analyze differences between controls and treated samples. Differences were considered statistically significant at P<0.05.

	Results

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Expression and Purification of r-apo(a) Derivatives
The r-apo(a) derivatives shown in Figure 1A were expressed in 293 (human embryonic kidney) cells. Proteins were purified to homogeneity from conditioned medium harvested from stably expressing cell lines by lysine-Sepharose affinity chromatography. Purified proteins (5 to 10 µg) were analyzed by SDS-PAGE under nonreducing conditions,³⁷ followed by Coomassie blue staining (Figure 1B); details of the analysis are given in the legend of Figure 1.

Platelet Function Studies
Neither the r-apo(a) derivatives, plasminogen, Lp(a), nor LDL, on their own, resulted in detectable platelet activation. Several concentrations of each substance were tested; the highest concentrations were 0.7 µmol/L r-apo(a) derivatives, 1.5 µmol/L plasminogen, 0.1 µmol/L Lp(a), and 0.3 µmol/L LDL. When stirred with suspensions of washed platelets in an aggregometer for up to 5 minutes, they did not stimulate shape change or aggregation, nor did they stimulate secretion of [¹⁴C]serotonin from prelabeled platelets over background levels (data not shown).

ADP-Induced Aggregation
Suspensions of washed platelets were stimulated with ADP in the absence or presence of the r-apo(a) derivatives shown in Figure 1A. In accord with our earlier observations,^{38 39} ADP stimulated only a primary phase of aggregation; secretion of [¹⁴C]serotonin was negligible. This primary, ADP-induced aggregation was not affected by the different r-apo(a) species. Several concentrations were tested, the highest being 0.7 µmol/L (Figure 2). Plasminogen, Lp(a), and LDL also had no effect on ADP-induced aggregation. Several concentrations of each substance were tested, the highest being 1.5 µmol/L plasminogen, 0.1 µmol/L Lp(a), and 0.3 µmol/L LDL (data not shown).

View larger version (13K): [in this window] [in a new window]   Figure 2. Lack of effect of r-apo(a) species (0.7 µmol/L) differing in the number of kringle IV type 2 domains on primary platelet aggregation stimulated by 4 µmol/L ADP in the presence of 100 µg/mL fibrinogen. Values are mean±SEM (n=3).

SFLLRN-Induced Responses
SFLLRN corresponds to the new amino terminus of the moderate-affinity thrombin receptor, protease-activated receptor-1 (PAR-1) after cleavage by thrombin; a synthetic peptide of this sequence stimulates platelet aggregation, secretion of granule contents, and formation of TXA₂.^{40 41 42 43 44} The concentration of SFLLRN (7.5 µmol/L) was chosen because, by itself, it caused considerably less than maximum aggregation and secretion of [¹⁴C]serotonin, making it possible to demonstrate whether enhancement or inhibition of these platelet responses occurred in the presence of the substances under investigation. Aggregation and secretion of [¹⁴C]serotonin stimulated by 7.5 µmol/L SFLLRN were significantly enhanced by 0.7 µmol/L 17K r-apo(a) (Figure 3A and 3B); TXB₂ (an index of TXA₂ formation) was also significantly increased (from 6.8±1.4 to 29.0±4.7 ng/10⁹ platelets, n=3, P<0.025). A lower concentration (0.35 µmol/L) of the 17K r-apo(a) also enhanced SFLLRN-induced responses (Figure 3A and B). Decreasing the number of kringle IV type 2 motifs in the r-apo(a) did not affect the enhanced responses to SFLLRN (Figure 3A and 3B). The lowest concentration of r-apo(a) that significantly enhanced SFLLRN-induced platelet aggregation was 0.175 µmol/L, with aggregation being increased from 14.6±7.1% to 30.8±7.5% and secretion of [¹⁴C]serotonin from 9.2±3.8% to 18.3±5.2% by 17K r-apo(a) (n=3; P<0.0025 and 0.025, respectively).

View larger version (26K): [in this window] [in a new window]   Figure 3. Enhancement by r-apo(a) species (0.35 or 0.7 µmol/L) differing in the number of kringle IV type 2 domains of (A) platelet aggregation and (B) secretion of [14C]serotonin stimulated by 7.5 µmol/L SFLLRN. Values are mean±SEM (n=3). *P<0.04 to 0.0075 (A) and <0.04 to 0.015 (B) compared with control.

Plasminogen, at concentrations of 0.7 and 1.5 µmol/L, also enhanced SFLLRN-induced aggregation and secretion, but not to the same extent as r-apo(a) (Figure 4). The combination of plasminogen (1.5 µmol/L) with 17K r-apo(a) (0.35 or 0.7 µmol/L) had an effect similar to that of 17K r-apo(a) alone. Lp(a) (0.025 to 0.1 µmol/L) greatly enhanced SFLLRN-induced aggregation and secretion of [¹⁴C]serotonin in a dose-dependent manner, and LDL (in the same concentration range) had a slight enhancing effect (Figure 5).

View larger version (22K): [in this window] [in a new window]   Figure 4. Enhancement by plasminogen (plgn) and the 12K r-apo(a) derivative of platelet responses stimulated by 7.5 µmol/L SFLLRN. Addition of SFLLRN is indicated by the arrow; the percentage of [14C]serotonin secretion from prelabeled platelets is given in the boxes beside the aggregation curves. L.T. indicates light transmission. Data are representative of 2 experiments with similar results.

View larger version (15K): [in this window] [in a new window]   Figure 5. Effects of purified Lp(a) (, ) and LDL (, ) on aggregation (, ) and secretion of [14C]serotonin (, ) when platelets were stimulated by 7.5 µmol/L SFLLRN. Data, expressed as percentages, are representative of 2 experiments with similar results.

	Discussion

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Although a number of case-control and prospective studies have demonstrated a correlation between elevated Lp(a) levels and atherosclerosis,^{1 2} the mechanisms underlying these observations remain unclear. Few studies have examined the effect of Lp(a) on platelet function, which may play a key role in the atherosclerotic process by enhancing intravascular thrombosis. We examined the effect of both Lp(a) and the unique protein component apo(a) on platelet aggregation induced by ADP and the thrombin receptor–activating peptide SFLLRN.

Our results show that plasminogen (up to 1.5 µmol/L), Lp(a) (up to 0.1 µmol/L), and LDL (up to 0.3 µmol/L) had no effect on the primary phase of aggregation stimulated by ADP; secretion of [¹⁴C]serotonin was negligible in all cases. Additionally, the r-apo(a) derivatives 6K, 12K, and 17K (containing various numbers of the major repeat kringle sequence; see Figure 1A) had no effect on this process when protein concentrations up to 0.7 µmol/L were used. This is in keeping with the results of Gries et al,¹⁶ who reported that Lp(a) had no significant effect on (maximum) aggregation induced by ADP. However, Pedreno et al¹⁷ reported that Lp(a) inhibited ADP-induced aggregation and that LDL enhanced it. The differences in these results may be related to the source of lipoprotein and methods used for lipoprotein purification.

In contrast with our results showing a lack of effect on ADP-induced aggregation, we observed enhancement of SFLLRN-induced platelet aggregation and concomitant secretion of granule contents in the presence of the 6K, 12K, and 17K r-apo(a) derivatives (at concentrations of 0.35 and 0.7 µmol/L for each r-apo(a) species); the Lp(a) particle (at concentrations of 0.025 to 0.1 µmol/L) also enhanced SFLLRN-induced responses in a dose-dependent manner. Given that LDL had only a slight enhancing effect (Figure 5), our data suggest that the potentiating effect of Lp(a) on SFLLRN-induced platelet responses is mediated by the apo(a) component of the Lp(a) particle. This is supported by the recent observation of Pedreno et al¹⁷ that LDL interacts with platelets through a different receptor than Lp(a), which may bind to platelets primarily via GP IIb of the GP IIb-IIIa complex.¹⁵ A potentiating effect of plasminogen on SFLLRN-induced aggregation was also observed; however, the enhancement of aggregation with the use of 0.7 µmol/L plasminogen was <50% of that observed using an equimolar concentration of the 12K r-apo(a) derivative (see Figure 4). As such, it appears that a major proportion of the observed potentiating effect of apo(a) on platelet aggregation arises as a result of the unique properties of apo(a) rather than those properties of the protein that are shared with plasminogen.

We recently showed that plasminogen binds to r-apo(a) or Lp(a) in solution, with K_d values of 20 and 6 nmol/L, respectively.⁴⁵ The complexes bind poorly to plasminogen-binding sites on fibrin. Accordingly, we investigated the effect of plasminogen on the r-apo(a)–mediated enhancement of platelet responses to SFLLRN. The effect of plasminogen and r-apo(a) in complex was identical to the effects of r-apo(a) alone, indicating that at their approximate physiological concentrations, plasminogen does not significantly influence the ability of r-apo(a) to enhance platelet responses to SFLLRN.

It is interesting to note that the effects of r-apo(a) on stimulated platelets are specific and contrasting with respect to mechanisms of action. For example, primary ADP-induced aggregation, which is not influenced by r-apo(a), does not involve the activation of intracellular phospholipase C,⁴⁶ whereas SFLLRN-induced responses, which are enhanced by both r-apo(a) and Lp(a), involve activation of phospholipase C, leading to hydrolysis of phosphatidylinositol 4,5-bisphosphate.^{47 48} Stimulation of platelets with thrombin also activates phospholipase C, and Gries et al¹⁶ reported that maximal platelet aggregation stimulated by 0.1 U/mL thrombin was not affected by Lp(a). However, it is not possible to observe an enhancement of a response that is already maximal. Thrombin induces minimal to maximal aggregation responses over a very narrow range of concentrations; thus, in the current study, SFLLRN, which does not have as steep a dose-response curve as thrombin, was used at a concentration that consistently stimulated weak platelet responses.

The concentrations of r-apo(a) derivatives used in our study (0.35 and 0.7 µmol/L) are physiologically relevant and correspond to plasma Lp(a) concentrations of 30 and 60 mg/dL for the 17K derivative, respectively; 30 mg/dL Lp(a) is above an apparent coronary risk threshold for Lp(a), which was defined on the basis of extensive epidemiological data.^{1 2}

The in vivo significance of the enhancement of SFLLRN-induced platelet responses by apo(a)/Lp(a) remains to be elucidated. However, it is possible that the potentiating effect of Lp(a) on platelet aggregation at the site of thrombus formation in vivo may contribute to thromboembolic complications associated with atherosclerosis. The proaggregatory effects of other proatherogenic lipoproteins (VLDL and LDL) on platelet function have been previously reported^{18 19 49} ; LDL has been shown to potentiate collagen-mediated platelet aggregation.¹⁶

We did not observe an effect of the number of kringle IV type 2 sequences on the magnitude of the potentiating effect of r-apo(a) on SFLLRN-mediated platelet responses. This suggests that in vivo, apo(a)/Lp(a) isoform size heterogeneity may not play a significant role in this process. In general, the significance of Lp(a) isoform size heterogeneity in the pathophysiological role of this lipoprotein remains undetermined, although Wild et al⁵⁰ recently reported an increased frequency of small apo(a) isoforms in men with myocardial infarction or coronary death in a case-control study. However, few Lp(a)-substrate interactions identified and characterized to date are dependent on apo(a) isoform size,² although there are several reports that apo(a) isoform size affects the binding of Lp(a) to fibrin and affects the extent to which Lp(a) inhibits fibrinolysis in vitro.^{51 52}

In summary, we demonstrated, for the first time, that the apo(a) component of Lp(a) potentiates SFLLRN-stimulated platelet responses in vitro. These observations suggest a novel mechanism by which Lp(a) may contribute to the thromboembolic complications of atherosclerosis.

	Acknowledgments

 
This work was supported by grants T2651 (M.L.R.) and T3104 (M.L.K.) from the Heart and Stroke Foundation of Ontario, Canada, and by grant HL-30086 (S.M.M.) from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md. Dr W. Sangrar held a Studentship Award and Dr M.L. Koschinsky holds a Research Scholarship from the Heart and Stroke Foundation of Canada.

	Footnotes

 
Presented in part at the 37th Annual Meeting of the American Society of Hematology, Seattle, Wash, December 1995.

Received September 23, 1997; accepted March 18, 1998.

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