L-4F Alters Hyperlipidemic (But Not Healthy) Mouse Plasma to Reduce Platelet Aggregation
Background and Purpose— Hyperlipidemia is associated with platelet hyperreactivity. We hypothesized that L-4F, an apolipoprotein A-I mimetic peptide, would inhibit platelet aggregation in hyperlipidemic mice.
Methods and Results— Injecting L-4F into apolipoprotein E (apoE)–null and low-density lipoprotein receptor–null mice resulted in a significant reduction in platelet aggregation in response to agonists; however, there was no reduction in platelet aggregation after injection of L-4F into wild-type (WT) mice. Consistent with these results, injection of L-4F into apoE-null mice prolonged bleeding time; the same result was not found in WT mice. Incubating L-4F in vitro with apoE-null platelet-rich plasma also resulted in decreased platelet aggregation. However, incubating washed platelets from either apoE-null or WT mice with L-4F did not alter aggregation. Compared with WT mice, unstimulated platelets from apoE-null mice contained significantly more 12-hydroxy 5,8,10,14-eicosatetraenoic acid, thromboxane A2, and prostaglandins D2 and E2. In response to agonists, platelets from L-4F–treated apoE-null mice formed significantly less thromboxane A2, prostaglandins D2 and E2, and 12-hydroxy 5,8,10,14-eicosatetraenoic acid.
Conclusion— By binding plasma-oxidized lipids that cause platelet hyperreactivity in hyperlipidemic mice, L-4F decreases platelet aggregation.
Hyperlipidemia is a risk factor associated with oxidative stress, the generation of oxidized lipoproteins, platelet hyperreactivity, and thrombogenic potential.1,2 Increasing evidence indicates that interactions between platelets and oxidized lipoproteins play a major role in the initiation, development, and progression of atherosclerosis.3–5 Apolipoprotein A-I (ApoA-I),6 apoA-IMilano, 7 or high-density lipoprotein (HDL)8 has been shown to inhibit platelet hyperreactivity and reverse the prothrombotic effects of hyperlipidemia. The atheroprotective and antithrombotic activity of HDL is generally attributed to the ability of HDL to promote reverse cholesterol transport and to the antioxidant and anti-inflammatory properties of the lipids and proteins associated with HDL.9 We recently reported10 that the apoA-I mimetic 4F peptides bind oxidized lipids with much higher affinity than apoA-I, thus explaining their ability to be effective in animal models and in preliminary human studies in vivo or in vitro at very low concentrations.
Patients with hyperlipoproteinemia show enhanced platelet reactivity, increased production of the arachidonic acid (AA) metabolites thromboxane A2 (TXA2) and 12-hydroxyeicosatetraenoic acid (12-HETE), secretion of mediators from platelet-dense granules, and a reduction of prostacyclin receptors on the platelet membrane that might prevent the antiplatelet action of prostacyclin.11,12
Based on the reported evidence on the role of HDL in platelet activation in hyperlipidemia and the knowledge that hyperlipidemic mice have elevated levels of oxidized lipids,13 we hypothesized that L-4F would exert an inhibitory effect on increased platelet aggregability in hyperlipidemic mice.
The apoA-I mimetic peptide L-4F was synthesized as previously described.14 Supplemental data (available at: http://atvb.ahajournals.org) describe other materials used in this study. Data are given as mean±SEM unless otherwise indicated.
Details on the mice used in these studies can also be found in the supplemental data. The mice were injected subcutaneously with L-4F at doses ranging between 0.01 and 1.00 mg/kg per day in 0.1 mL of vehicle (ABCT) containing 50-mmol/L ammonium bicarbonate, pH 7.0, and 0.1-mg/mL Tween-20 or vehicle only for various time periods; one hour after the last injection, blood was collected. All animal procedures were approved by the University of California, Los Angeles, Animal Research Committee.
Platelet Preparation and Aggregation
Blood was collected in 3.8% sodium citrate (9:1 vol/vol) from the retro-orbital plexus of isoflurane-anesthetized mice using heparin-coated capillary tubes. Blood from 8 to 10 mice was pooled for the experiments. The citrated blood was centrifuged at 150g for 10 minutes at room temperature to obtain platelet-rich plasma (PRP) and centrifuged again at 1800g for 15 minutes to obtain platelet-poor plasma.15 Contaminating red blood cells and leukocytes in the PRP were removed by a two-minute centrifugation at 180g. The number of platelets in PRP was counted manually by light microscopy using a hemocytometer at a magnification of ×400.16 Five PRP samples were counted by one investigator and recounted by two additional observers blinded to treatment (B.Y. and S.C.). The coefficient of variation for interobserver measurements was 12%±1%. The platelet number in the PRP was adjusted to 1×108 to 5×108 cells per milliliter, with platelet-poor plasma as a diluent. For the preparation of washed platelets, blood collected in acid-citrate-dextrose was processed as described without any modifications.17 Briefly, PRP containing prostaglandin E1, 1 μg/mL, to prevent activation during washing was sedimented by centrifugation at 1800g for ten minutes and gently washed twice with the platelet wash buffer (pH 6.5). After centrifugation, the platelet pellet was resuspended in a modified calcium-free HEPES-Tyrode buffer (pH 7.4) and diluted to the final concentration of 1×108 to 3×108 platelets per milliliter.17 Platelets suspended in either plasma or HEPES-Tyrode buffer were incubated at room temperature for 30 minutes with gentle agitation. Murine fibrinogen, 1 mg/mL, and calcium chloride, 1 mmol/L, were added to the washed platelet suspension 30 seconds before agonist addition. Platelet aggregation was conducted at 37°C at a constant stirring rate of 1000 rpm in a four-channel profiler (PAP-4 Platelet Aggregation Profiler; Bio/Data Corporation, Horsham, Pa). Aggregation of platelets was elicited by the addition of the following agonists: adenosine diphosphate (ADP), 0.5 to 20.0 μM; collagen, 0.048 to 0.190 mg/mL; and AA, 25 to 500 μg/mL. Suboptimal concentrations of agonists were used after establishing the concentrations that caused minimal and maximal aggregation for each experiment.18 All experiments were repeated at least three times using platelets from different mice. The resulting aggregation, measured as the change in light transmission, was recorded until a plateau was reached. The following platelet aggregation parameters were used to evaluate the effects of L-4F on platelets: amplitude or the extent of aggregation represented by the total decrease in the optical density and expressed as percentage aggregation; the slope or Vmax (ie, the maximum sustained rate of aggregation) determined from the steepest slope of the aggregation curve and expressed as a change in the optical density per second; and lag time (for PRP only), representing the elapsed time (in seconds) between agonist addition and the start of aggregation.19,20
For in vitro studies, L-4F (0.01, 0.10, and 1.00 μg/mL), or vehicle alone, was added to PRP or to washed platelets and incubated for 60 minutes at 37°C with gentle stirring in the aggregometer; platelet aggregation was determined in response to the indicated agonists.
Measurement of TXA2 Formation
Mice were injected subcutaneously with L-4F at a dose of 1 mg/kg per day in 0.1 mL of vehicle (ABCT) or vehicle only on day one and were injected twice more every 24 hours. One hour after the last injection, blood was collected for platelet preparation and aggregation studies. The formation of TXA2 in PRP containing 1×108 platelets per milliliter from L-4F– or vehicle-injected mice was measured by determining thromboxane B2 (TXB2), the stable metabolite of TXA2 in the samples used for platelet aggregation in the presence or absence of the cyclooxygenase (COX) 1 inhibitor, SC-560 (1 μmol/L), which was added in dimethyl sulfoxide and preincubated with the PRP for 30 minutes at room temperature. The reaction initiated by the addition of ADP (20 μmol/L), collagen (0.19 mg/mL), or AA (500 μg/mL) for 5 minutes at 37°C at a stirring rate of 1000 rpm was terminated by the addition of 80 μmol/L of aspirin and 10 mmol/L of EDTA, followed by rapid freezing and storing at −80°C.20 The amount of TXB2 in medium was determined by using the TXB2 enzyme immunoassay kit, according to the procedure described by the manufacturer (Assay Designs, Inc, Ann Arbor, Mich).
LC-MS/MS analysis is described in the supplemental material (available at: http://atvb.ahajournals.org).
Mice were injected subcutaneously with L-4F at a dose of 1 mg/kg per day in 0.1 mL of vehicle or vehicle only on day one and were injected twice more every 24 hours. One hour after the last injection, tail bleeding time was measured in isoflurane-anesthetized mice. Aspirin, 10 mg/kg, administered by stomach gavage 24 hours before bleeding, was used as a positive control. For details of the method used to measure tail bleeding time, please see the supplemental material (available at: http://atvb.ahajournals.org).
Plasma lipoprotein and lipid levels were determined as described previously.21
For methods used for statistical analyses, please see supplemental material (available at: http://atvb.ahajournals.org). Differences were considered statistically significant at P<0.05 or less.
L-4F Inhibits Agonist-Induced Platelet Aggregation Ex Vivo in Apolipoprotein E– and Low-Density Lipoprotein Receptor–Null Mice
L-4F (0.01, 0.1 and 1 mg/kg) injected subcutaneously dose-dependently inhibited the ex vivo aggregation of platelets (Figure 1) stimulated with: (a) ADP (IC50 values of 2.6, 6.69, 7.32** and 7.56* mM for administration of ABCT or L-4F at doses of 0.01, 0.1, and 1 mg/kg respectively; *P<0.05; **P<0.01); (b) collagen (IC50 values of 0.0035, 0.013, 0.049 and 0.082* mg/mL respectively); (c) AA with IC50 values of 91.4, 95.6, 551** and 685** mg/mL respectively. In addition, L-4F significantly reduced the slope and increased the lag time in response to AA, collagen and ADP (data not shown).
Similar results were obtained when L-4F (but not vehicle) was injected into low-density lipoprotein receptor (LDLR)–null mice fed an atherogenic diet. Because of the marked hyperlipidemia associated with turbid plasma that was induced in these mice, it was necessary to use washed platelet suspensions rather than PRP. Aggregation elicited by collagen, 0.096 mg/mL, was significantly decreased (P=0.003) in the washed platelet suspensions from L-4F–injected LDLR-null mice (72.00%±3.61% aggregation) but not in those from vehicle-injected LDLR-null mice (101.33%±2.85% aggregation). AA (250 μg/mL) induced aggregation was also significantly inhibited (P=0.006) in the washed platelet suspensions from L-4F–injected LDLR-null mice (52.33%±5.89% aggregation) but was not significantly inhibited in the platelets from vehicle-injected LDLR-null mice (89.33%±3.67% aggregation).
L-4F Inhibits Agonist-Induced Platelet Aggregation In Vitro in the Presence of Plasma From Apolipoprotein E–Null Mice But Not in the Absence of Plasma
The incubation of PRP from apolipoprotein E (apoE)–null mice with L-4F, 500 ng/mL (but not vehicle), significantly reduced platelet aggregation in response to ADP (Figure 2, panel A) and collagen (Figure 2, panel B). In contrast to these results, adding L-4F in vitro to washed platelets from either apoE-null (Figure 3, panels A-C) or wild-type (WT) mice did not inhibit platelet aggregation (Figure 3, panels D-F).
These results indicate that L-4F does not have a direct effect on platelets but acts through some component of plasma that influences platelet function. As previously reported,22 there was no change in plasma total cholesterol, triglycerides, HDL cholesterol, or apolipoprotein B–containing cholesterol levels after the injection of L-4F in these experiments (data not shown).
A single subcutaneous injection of L-4F in apoE-null mice significantly inhibited platelet aggregation in response to ADP, collagen, or AA for up to 72 hours after injection (data not shown). Ninty-six hours after injection, there was no significant difference in platelet aggregation between mice injected with L-4F or vehicle alone in response to any of the agonists (data not shown).
Although a single subcutaneous injection of L-4F, 0.01 mg/kg per day, was without a significant effect (Figure 1), the administration of the peptide by Alzet osmotic pumps delivering L-4F at a dose of 0.01 mg/kg per day for two weeks significantly reduced platelet aggregation in response to ADP, collagen, or AA compared with mice implanted with pumps delivering the same amount of vehicle without L-4F (data not shown).
L-4F Inhibits Agonist-Induced TXB2 Production
TXB2 is the stable hydrolysate of TXA2, which is generated from the AA released from platelet plasma membranes after the sequential activities of the cytosolic phospholipase A2–COX-1–TXA2 synthase pathway.23 After treatment with L-4F, platelet stimulation with AA resulted in significantly less TXB2 production (approximately 30% less) (data not shown). Under these conditions in the same samples, aggregation was reduced by more than 40% (data not shown). The addition of the COX-1 inhibitor SC-560 further inhibited platelet TXB2 formation and aggregation in platelets taken from both vehicle- and L-4F–treated mice (data not shown). Similarly, the slope exhibited significant reductions that were comparable with the reduction in TXB2 production (data not shown). Conversely, the lag times were increased in the platelets collected from the L-4F–injected mice, and the addition of SC-560 to these platelets further increased the lag time; however, this did not reach statistical significance. Preincubation of platelets from the vehicle-treated mice with SC-560 significantly prolonged the lag time (data not shown). Similar results were obtained when the platelets were stimulated with collagen (data not shown). In the platelets stimulated with ADP, the addition of SC-560 did not significantly inhibit these parameters beyond that achieved with L-4F, suggesting that L-4F may inhibit the same segment of the AA–COX-1–TXA2 pathway that is antagonized by SC-560 under these conditions (data not shown).
L-4F Treatment Inhibits Agonist-Induced Formation of TXB2, Prostaglandins D2 and E2, and 12-HETE Without Changing the Concentration of Plasma Lipids
In addition to TXB2, a number of other eicosanoids derived from the AA cascade via COX-1 and 12-lipoxygenase enzymatic pathways were analyzed by LC-MS/MS. PRP obtained from L-4F– or vehicle-treated mice was stimulated with the following agonists: collagen (0.05 mg/mL), ADP (20 μmol/L), AA (125 μg/mL), calcium ionophore A23187 (2.5 μmol/L), and the TXA2 mimetic (U44619) (1 μmol/L).24
As shown in Table 1, except for U44619, which directly activates platelet TXA2 receptors,24 the production of AA metabolites 12-HETE, TXB2, and prostaglandins D2 (PGD2) and E2 (PGE2) was significantly diminished in platelets from L-4F–treated mice vs vehicle controls. The rank order for AA metabolites (calculated as nanograms per 3×108 platelets) was 12-HETE > TXA2 > PGE2 > PGD2; the rank order for AA metabolites (calculated as a percentage of the most abundant metabolite) was 12-HETE (100%) > TXA2 (12%) > PGE2 (0.6%) > PGD2 (0.3%). In stimulated platelet suspensions preincubated with SC-560, the production of TXB2 was significantly reduced in an agonist-dependent manner with the potency rank order of AA > collagen > A23187 > U46619 > ADP, and was associated with a marked increase of 12-HETE formation (Table 2), suggesting that in the presence of COX-1 inhibitor the AA is diverted toward the 12-lipoxygenase pathway to generate additional 12-HETE.25
AA Metabolite Levels Are Higher in Platelets From ApoE-Null Mice Than in Platelets From WT Mice
Unstimulated platelets from apoE-null mice contained significantly more 12-HETE, TXB2, PGD2, and PGE2 than unstimulated platelets from WT C57BL/6 mice (Table 3).
L-4F Treatment Results in Prolonged Bleeding Time in ApoE-Null Mice But Not in WT Mice
The treatment of apoE-null mice with L-4F resulted in a significant (P<0.001) increase in the bleeding time compared with vehicle-treated mice (3.76±0.73 vs 1.68±0.25 minutes). The addition of oral aspirin significantly (P<0.01) prolonged the bleeding time of vehicle-treated apoE-null mice (from 1.68±0.25 to 4.65±0.65 minutes). However, in L-4F–treated apoE-null mice, the increase in bleeding time after L-4F treatment alone (3.76±0.73 minutes) was not significantly increased by the addition of aspirin (4.14±0.48 minutes). WT C57BL/6 mice did not show a significant increase in bleeding time with L-4F treatment (2.06±0.24 minutes) compared with vehicle treatment alone (1.84±0.21 minutes). However, the bleeding time in response to aspirin in WT mice increased significantly (P<0.01) to 3.93±0.55 minutes. Untreated (ie, no injections) WT C57BL/6 mice had a slightly, but significantly, longer bleeding time (2.10±0.21 minutes) than untreated apoE-null mice (1.62±0.10 minutes) (P=0.049).
In the present study, we investigated the effects of the apoA-I mimetic peptide L-4F on platelet aggregation in WT, LDLR-null, and apoE-null mice on the same genetic background. Because no information regarding the effects of apoA-I mimetic peptides on platelet function was available, we chose to use several physiologically active agonists (collagen, ADP, AA, A23187, and U46619) with distinct platelet activation and aggregation signaling pathways.
The administration of L-4F in vivo significantly reduced ex vivo the percentage of aggregating platelets (Figure 1), accompanied by the expected changes in slope and lag time (data not shown) in response to increasing concentrations of ADP, AA, or collagen compared with vehicle-treated mice. L-4F treatment in vitro of PRP produced similar results; however, incubation of washed platelets with L-4F was without effect, suggesting that L-4F acts on plasma components that modulate platelet function.
The plasma half-life of L-4F after a single subcutaneous injection in mice is on the order of only one hour.26 The fact that a dose of 0.01 mg/kg of L-4F administered by a single subcutaneous injection was not effective (Figure 1), but that the same dose administered by continuous infusion by Alzet pumps for two weeks was effective (data not shown), suggests that the time during which the peptide is present in plasma may be more important than the peak plasma concentration.
TXA2, one of the major metabolites of AA and a potent endogenous platelet agonist and vasoconstrictor, plays an important role in platelet-rich thrombus formation.27,28 TXA2 is produced in platelets from AA via the COX-TXA2 synthase pathway.29 The stable hydrolysate of TXA2, TXB2, was significantly reduced in agonist-stimulated platelets obtained from L-4F–treated mice compared with vehicle-treated mice. The inhibition of TXB2 formation paralleled the effects observed for platelet aggregation, suggesting that the inhibition of the COX-TXA2 synthase pathway was at least in part responsible for the reduction in platelet aggregation.
The formation of TXB2, PGD2, PGE2, and 12-HETE was significantly inhibited in platelets obtained from L-4F–injected mice and stimulated with collagen, A23187, ADP, or AA but not with U46619. U46619 addition in the absence of COX-1 inhibition continues the cycle of TXA2 receptor activation–AA liberation–TXA2 formation, generating an excess of TXA2 (Table 1). The inhibition of COX-1 further reduced TXB2 (Table 2) and PDG2 and PGE2 formation (data not shown) and significantly increased 12-HETE accumulation (Table 2), confirming that AA used for both 12-HETE and TXB2 biosynthesis is derived from a single phospholipid pool that can be shunted from one pathway to the other.25 In addition, the use of U46619 in the presence of COX-1 blockade also demonstrated that L-4F lacks any inhibitory effect on TXA2 synthase activity or on the TXA2 receptor (Table 2).
The decreased platelet aggregation and TXB2 formation after treatment with L-4F was associated with an increased bleeding time in the apoE-null mice but not in the WT C57BL/6 mice. Although the difference in bleeding time between the two treatment groups was statistically significant, no spontaneous bleeding was observed in the L-4F–injected mice and no additive or synergistic effects were observed between aspirin and L-4F. The small differences in the bleeding times in vehicle-treated mice vs untreated mice may indicate a slight effect of the vehicle.
Our results indicate that L-4F decreases the hypersensitivity of platelets to agonist stimulation in hyperlipidemic mice but not in normolipidemic C57BL/6 mice. L-4F does not have a direct effect on platelets but appears to work through a plasma component, as previously noted. Forte et al13 reported that the type of oxidized lipids that have been shown to have a particularly high affinity for the 4F peptide10 are significantly increased in the plasma of apoE-null mice compared with WT mice. Our results are consistent with L-4F binding and the removal of these oxidized lipids from plasma in hyperlipidemic mice, resulting in altered platelet function. The precise identity and mechanism(s) by which such oxidized lipids influence platelet function will require further studies.
Sources of Funding
This study was supported in part by grants HL-30568 and HL-34343 from the US Public Health Service; and the Laubisch, Castera, and M. K. Grey Funds at the University of California, Los Angeles.
Drs Navab, Reddy, Anantharamaiah, and Fogelman are principals in Bruin Pharma; and Dr Fogelman is an officer in Bruin Pharma.
Received July 6, 2009; revision accepted November 19, 2009.
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