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Potent reduction of plasma lipoprotein (a) with an antisense oligonucleotide in human subjects does not affect ex vivo fibrinolysis

Open AccessPublished:September 24, 2019DOI:https://doi.org/10.1194/jlr.P094763
      It is postulated that lipoprotein (a) [Lp(a)] inhibits fibrinolysis, but this hypothesis has not been tested in humans due to the lack of specific Lp(a) lowering agents. Patients with elevated Lp(a) were randomized to antisense oligonucleotide [IONIS-APO(a)Rx] directed to apo(a) (n = 7) or placebo (n = 10). Ex vivo plasma lysis times and antigen concentrations of plasminogen, factor XI, plasminogen activator inhibitor 1, thrombin activatable fibrinolysis inhibitor, and fibrinogen at baseline, day 85/92/99 (peak drug effect), and day 190 (3 months off drug) were measured. The mean ± SD baseline Lp(a) levels were 477.3 ± 55.9 nmol/l in IONIS-APO(a)Rx and 362.1 ± 89.9 nmol/l in placebo. The mean± SD percentage change in Lp(a) for IONIS-APO(a)Rx was −69.3 ± 12.2% versus −5.4 ± 6.9% placebo (P < 0.0010) at day 85/92/99 and −15.6 ± 8.9% versus 3.2 ± 12.2% (P = 0.003) at day 190. Clot lysis times and coagulation/fibrinolysis-related biomarkers showed no significant differences between IONIS-APO(a)Rx and placebo at all time points. Clot lysis times were not affected by exogenously added Lp(a) at concentrations up to 200 nmol/l to plasma with very low (12.5 nmol/l) Lp(a) levels, whereas recombinant apo(a) had a potent antifibrinolytic effect. In conclusion, potent reductions of Lp(a) in patients with highly elevated Lp(a) levels do not affect ex vivo measures of fibrinolysis; the relevance of any putative antifibrinolytic effects of Lp(a) in vivo needs further study.
      Lipoprotein(a) [Lp(a)] is a genetic risk factor for CVD and calcific aortic valve stenosis. Lp(a) is thought to mediate clinical events by three main mechanisms: proatherogenic effects via its LDL cholesterol (LDL-C) moiety (
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      Lipoprotein (a): truly a direct prothrombotic factor in cardiovascular disease?.
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      Lp(a) has high homology (75–99%) to plasminogen but lacks protease activity and therefore has been hypothesized to inhibit fibrinolysis and mediate prothrombotic potential. This hypothesis has been supported by in vitro/ex vivo studies primarily using free apo(a), of which little if any is present in plasma in vivo, rather than purified Lp(a) (
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      ). Furthermore, elevated Lp(a) levels are causally associated with calcific aortic valve stenosis, where thrombosis is not a part of the clinical phenotype (
      • Tsimikas S.
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      ). On the other hand, whether Lp(a) might directly contribute to the thrombotic sequelae of arterial plaque rupture has been difficult to assess because of the inability to disentangle such effects from underlying atherosclerotic disease.
      The role of Lp(a) lowering on its effects on fibrinolysis in humans has not been previously investigated due to the lack of specific Lp(a) lowering agents. IONIS-APO(a)Rx and IONIS-APO(a)-LRx, having the same sequence as IONIS-APO(a)Rx but additionally containing the hepatocyte targeting moiety n-acetylgalactosamine, are second-generation antisense oligonucleotides directed to apo(a). In phase I and II clinical trials they have been demonstrated to lower mean Lp(a) levels by 70% to 92% (
      • Tsimikas S.
      • Viney N.J.
      • Hughes S.G.
      • Singleton W.
      • Graham M.J.
      • Baker B.F.
      • Burkey J.L.
      • Yang Q.
      • Marcovina S.M.
      • Geary R.S.
      • et al.
      Antisense therapy targeting apolipoprotein(a): a randomised, double-blind, placebo-controlled phase 1 study.
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      • Viney N.J.
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      • Xia S.
      • Tami J.A.
      • Yu R.Z.
      • Marcovina S.M.
      • Hughes S.G.
      • Graham M.J.
      • Crooke R.M.
      • et al.
      Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.
      ). This degree of reduction in Lp(a) is much higher than has been achieved by agents that are not specific for Lp(a) lowering such as niacin (
      • Superko H.R.
      • Zhao X.Q.
      • Hodis H.N.
      • Guyton J.R.
      Niacin and heart disease prevention: engraving its tombstone is a mistake.
      ) and antibody inhibitors of proprotein convertase subtilisin/kexin type 9 (
      • Stiekema L.C.A.
      • Stroes E.S.G.
      • Verweij S.L.
      • Kassahun H.
      • Chen L.
      • Wasserman S.M.
      • Sabatine M.S.
      • Mani V.
      • Fayad Z.A.
      Persistent arterial wall inflammation in patients with elevated lipoprotein(a) despite strong low-density lipoprotein cholesterol reduction by proprotein convertase subtilisin/kexin type 9 antibody treatment.
      ).
      In this study, we evaluated whether substantial lowering of elevated Lp(a) results in an improvement in fibrinolytic potential using ex vivo clot lysis assays and measuring coagulation/fibrinolysis biomarkers.

      MATERIALS AND METHODS

      Study subjects

      Study subjects were enrolled from three trials assessing antisense oligonucleotides directed to apo(a). The first study was a phase 1 study that used ISIS-APO(a)Rx in healthy volunteers (
      • Tsimikas S.
      • Viney N.J.
      • Hughes S.G.
      • Singleton W.
      • Graham M.J.
      • Baker B.F.
      • Burkey J.L.
      • Yang Q.
      • Marcovina S.M.
      • Geary R.S.
      • et al.
      Antisense therapy targeting apolipoprotein(a): a randomised, double-blind, placebo-controlled phase 1 study.
      ), the second study was a phase 2 study in subjects with elevated Lp(a) with the same drug [but relabeled as IONIS-APO(a)Rx] (
      • Viney N.J.
      • van Capelleveen J.C.
      • Geary R.S.
      • Xia S.
      • Tami J.A.
      • Yu R.Z.
      • Marcovina S.M.
      • Hughes S.G.
      • Graham M.J.
      • Crooke R.M.
      • et al.
      Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.
      ), and the third study was a phase 1 study in healthy volunteers with elevated Lp(a) that used IONIS-APO(a)LRx, an n-acetylgalactosamine-conjugated antisense oligonucleotide to apo(a) (
      • Viney N.J.
      • van Capelleveen J.C.
      • Geary R.S.
      • Xia S.
      • Tami J.A.
      • Yu R.Z.
      • Marcovina S.M.
      • Hughes S.G.
      • Graham M.J.
      • Crooke R.M.
      • et al.
      Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.
      ).
      Ex vivo clot lysis time was measured in baseline samples from healthy subjects (n = 16) enrolled in a phase 1 study of ISIS-APO(a)Rx(18) to ascertain the normal range of clot lysis times. Subjects were healthy adults aged 18–65 years with a BMI <32 kg/m2 and Lp(a) levels >25 nmol/l. In the phase 2 study, patients (n = 64) with Lp(a) >125 nmol/l (>50 mg/dl) were randomized to placebo or IONIS-APO(a)Rx, dosed subcutaneously at 100 mg weekly for 1 month, 200 mg weekly for the second month, and 300 mg weekly for the third month (ClinicalTrials.gov identifier: NCT02160899) (
      • Tsimikas S.
      • Viney N.J.
      • Hughes S.G.
      • Singleton W.
      • Graham M.J.
      • Baker B.F.
      • Burkey J.L.
      • Yang Q.
      • Marcovina S.M.
      • Geary R.S.
      • et al.
      Antisense therapy targeting apolipoprotein(a): a randomised, double-blind, placebo-controlled phase 1 study.
      ). In a subset of these patients with the highest Lp(a) levels [10 placebo, 7 IONIS-APO(a)Rx] ex vivo plasma clot lysis time and antigen concentrations of plasminogen, factor XI, plasminogen activator inhibitor 1 (PAI-1), thrombin activatable fibrinolysis inhibitor (TAFI), fibrinogen and oxidized phospholipid content on apoB-100, apo(a), and plasminogen (
      • Leibundgut G.
      • Arai K.
      • Orsoni A.
      • Yin H.
      • Scipione C.
      • Miller E.R.
      • Koschinsky M.L.
      • Chapman M.J.
      • Witztum J.L.
      • Tsimikas S.
      Oxidized phospholipids are present on plasminogen, affect fibrinolysis, and increase following acute myocardial infarction.
      ) were measured at baseline, day 85/92/99 (representing peak drug effect), and day 190 (3 months off drug). Blood sampling at day 85/92/99 was used, as not all patients had blood sampling on all 3 days. To assess whether the findings were drug-specific, ex vivo clot lysis assays in three otherwise healthy human volunteers with highly elevated Lp(a) levels enrolled in the IONIS-APO(a)LRx trial (ClinicalTrials.gov identifier: NCT02414594) (
      • Viney N.J.
      • van Capelleveen J.C.
      • Geary R.S.
      • Xia S.
      • Tami J.A.
      • Yu R.Z.
      • Marcovina S.M.
      • Hughes S.G.
      • Graham M.J.
      • Crooke R.M.
      • et al.
      Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.
      ). Approval for the study was obtained at local institutional review boards, and subjects gave written informed consent to participate.

      Ex vivo clot formation and clot lysis assays

      Ex vivo clot formation and clot lysis assays were performed in 33% diluted patient plasma collected in EDTA added to wells containing final concentrations of 10 mmol/l CaCl2, 0.01% thromboplastin-DS, 10 μmol/l phosphatidylserine-phosphatidylcholine (80:20) vesicles, and 1 nmol/l tissue-type plasminogen activator (tPA). Clot formation and clot lysis times were monitored turbidometrically at 37°C in a plate-reading spectrophotometer (Fig. 1). In some experiments, plasma from a single donor with low Lp(a) was used, and clots were supplemented with increasing doses of Lp(a), purified as described (
      • Bouchareb R.
      • Mahmut A.
      • Nsaibia M.J.
      • Boulanger M-C.
      • Dahou A.
      • Lépine J-L.
      • Laflamme M-H.
      • Hadji F.
      • Couture C.
      • Trahan S.
      • et al.
      Autotaxin derived from lipoprotein(a) and valve interstitial cells promotes inflammation and mineralization of the aortic valve.
      ), or the following recombinant variants of apo(a) were used: 17-kringle (17K), 17KΔLBS10, 30K, 14K, 12K, and 6K (
      • Feric N.T.
      • Boffa M.B.
      • Johnston S.M.
      • Koschinsky M.L.
      Apolipoprotein(a) inhibits the conversion of Glu-plasminogen to Lys-plasminogen: a novel mechanism for lipoprotein(a)-mediated inhibition of plasminogen activation.
      ).
      Figure thumbnail gr1
      Fig. 1.Methodology of clot lysis assay. Left: Design of the ex vivo clot lysis assay, where plasma in the presence or absence of purified Lp(a) or apo(a) is added to the wells of a 96-well plate containing microaliquots of tissue factor (TF) and CaCl2 to initiate clot formation and tPA to initiate fibrinolysis. Right: Representative lysis profile showing how clot time and lysis time are derived from the midpoints of the increase and decrease in clot turbidity, respectively. In all cases the clot time is subtracted from the lysis time.

      Laboratory analyses

      Lp(a) concentrations were measured in nmol/l, and apo(a) isoform sizes were measured at the Northwest Lipid Metabolism and Diabetes Research Laboratories at the University of Washington (Seattle, WA) as previously described (
      • Viney N.J.
      • van Capelleveen J.C.
      • Geary R.S.
      • Xia S.
      • Tami J.A.
      • Yu R.Z.
      • Marcovina S.M.
      • Hughes S.G.
      • Graham M.J.
      • Crooke R.M.
      • et al.
      Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.
      ). Plasma concentrations of apoB-100, total cholesterol, LDL-C, HDL cholesterol (HLD-C), VLDL cholesterol, and triglycerides were measured with commercially available assays (Medpace Reference Laboratories) (
      • Viney N.J.
      • van Capelleveen J.C.
      • Geary R.S.
      • Xia S.
      • Tami J.A.
      • Yu R.Z.
      • Marcovina S.M.
      • Hughes S.G.
      • Graham M.J.
      • Crooke R.M.
      • et al.
      Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.
      ). Lp(a) cholesterol was estimated by converting the Lp(a) particle number in nmol/l to Lp(a) in mg/dl and dividing by 2.4; this value was then multiplied by 0.3 (
      • Viney N.J.
      • van Capelleveen J.C.
      • Geary R.S.
      • Xia S.
      • Tami J.A.
      • Yu R.Z.
      • Marcovina S.M.
      • Hughes S.G.
      • Graham M.J.
      • Crooke R.M.
      • et al.
      Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.
      ). Antigen concentrations of Factor XI (Affinity Biologicals), fibrinogen (Enzo Life Sciences), PAI-1 (Life Technologies), and TAFI (Affinity Biologicals) were measured with commercial assays. Plasminogen, OxPL-apoB, OxPL-apo(a), and OxPL-plasminogen were measured with in-house assays at University of California San Diego as previously described (
      • Leibundgut G.
      • Arai K.
      • Orsoni A.
      • Yin H.
      • Scipione C.
      • Miller E.R.
      • Koschinsky M.L.
      • Chapman M.J.
      • Witztum J.L.
      • Tsimikas S.
      Oxidized phospholipids are present on plasminogen, affect fibrinolysis, and increase following acute myocardial infarction.
      ).

      Statistical analysis

      Continuous data are expressed as means ± SDs; the comparisons between the two treatment arms [IONIS-APO(a)Rx treatment and placebo] in the IONIS-APO(a)Rx trial were performed using the two-sample t-test. If data departed substantially from normality, the Wilcoxon rank-sum test was used. Categorical data are expressed as frequencies (number of patients in category) and percentages; the comparisons between the two treatment arms in the IONIS-APO(a)Rx trial were performed using Fisher's exact test. Correlations between lysis time and baseline plasma Lp(a) were assessed by Spearman's rank correlation coefficient, and differences in lysis time between Lp(a) and recombinant apo(a) variants were assessed by ANOVA with Lp(a)/apo(a) or apo(a) variants and their concentrations as the factors and adjusted using Tukey's post hoc analysis or Dunnett's multiple comparison test, respectively. The analyses were performed with SAS version 9.4 (SAS Institute).

      RESULTS

      Baseline characteristics of the groups and biomarkers of fibrinolysis and coagulation are shown in Table 1. The mean ± SD baseline Lp(a) levels in the placebo and IONIS-APO(a)Rx groups were 362.1 ± 89.9 nmol/l and 477.3 ± 55.9 nmol/l, respectively. The mean size of apo(a) isoforms was 16–17, and LPA SNPs r10455872 or rs3798820, which are associated with elevated Lp(a), were present in >80% of patients. The mean ± SD change in Lp(a) was −69.3 ± 12.2% versus −5.4 ± 6.9% (P < 0.0010) at day 85/92/99 and −15.6 ± 8.9% versus 3.2 ± 12.2% (P = 0.003) at day 190 in the IONIS-APO(a)Rx versus placebo, respectively. The IONIS-APO(a)-LRx group had baseline Lp(a) levels of 194.0 ± 52.8 nmol/l.
      TABLE 1.Baseline characteristics and coagulation/fibrinolysis parameters
      CharacteristicPlacebo (n = 10)IONIS-APO(a)Rx (n = 7)PIONIS-APO(a)-LRx (n = 3)
      Age (years)57 ± 1060 ± 90.44058 ± 9
      Gender (M:F)6:42:50.3351:2
      BMI (kg/m2)28.4 ± 3.728.9 ± 5.40.83522.4 ± 1.2
      Race [n (%)]1.00
      White9 (90.0)7 (100.0)2 (66.7)
      Black1 (10.0)0 (0.0)1 (33.3)
      Medical history
      Hypertension4 (40.0)2 (28.6)1.0000 (0.0)
      Diabetes mellitus0 (0.0)1 (14.3)0.4120 (0.0)
      Coronary artery disease4 (40.0)1 (14.3)0.3380 (0.0)
      Myocardial infarction4 (40.0)0 (0.0)0.1030 (0.0)
      Percutaneous coronary intervention or coronary-artery bypass grafting2 (20.0)1 (14.3)1.0000 (0.0)
      Stroke or transient ischemic attack0 (0.0)0 (0.0)NA0 (0.0)
      Carotid or peripheral artery disease0 (0.0)1 (14.3)0.4120 (0.0)
      Aortic stenosis0 (0.0)0 (0.0)NA0 (0.0)
      Lipid-lowering medications
      Statin8 (80.0)5 (71.4)0 (0.0)
      Ezetimibe4 (40.0)3 (42.9)0 (0.0)
      Other0 (0.0)0 (0.0)0 (0.0)
      Any lipid-modulating medication8 (80.0)5 (71.4)1.0000 (0.0)
      Lp(a) (nmol/l)
      Baseline362.1 ± 89.9477.3 ± 55.90.009194.0 ± 52.8
      End of treatment341.9 ± 87.2148.0.3 ± 64.7<0.00112.1 ± 4.9
      End of follow-up368.3 ± 73.5401.2 ± 50.10.32254.8 (44.7)
      Lp(a) size, major isoform (number of kringle repeats)16.7 ± 1.516.7 ± 0.80.91717.3 ± 1.5
      LPA snp group [n, %]
      rs3798220 homozygotes1 (10.0)0 (0.0)0 (0.0)
      rs10455872 homozygotes1 (10.0)1 (14.3)0 (0.0)
      rs3798220 and rs10455872 heterozygotes2 (20.0)2 (28.6)0 (0.0)
      heterozygous rs104558722 (20.0)1 (14.3)1 (33.3)
      heterozygous rs37982203 (30.0)2 (28.6)1 (33.3)
      Either rs3798220 or rs104558729 (90.0)6 (83.7)1.0002 (66.7)
      Estimated Lp(a)-C (mg/dl)45.3 ± 11.259.7 ± 7.00.00924.3 ± 6.6
      LDL-C (mg/dl)109.7 ± 36.8116.5 ± 39.50.773139.0 ± 35.1
      Corrected LDL-C64.4 ± 41.156.8 ± 40.20.962114.7 ± 41.1
      apoB (mg/dl)90.5 ± 18.696.9 ± 23.20.53595.2 ± 18.1
      Total cholesterol (mg/dl)190.1 ± 51.4192.6 ± 39.40.914217.3 ± 44.2
      HDL-C (mg/dl)55.5 ± 28.451.4 ± 9.70.72161.2 ± 17.8
      Triglycerides (mg/dl)125.6 ± 38.2123.3 ± 26.30.89285.5 ± 20.3
      OxPL-plasminogen (nM)160.6 ± 88.7134.3 ± 39.10.810102.8 ± 82.3
      OxPL-apoB (nM)27.2 ± 4.432.7 ± 3.00.01224.8 ± 7.0
      OxPL-apo(a) (nM)57.5 ± 13.963.9 ± 11.30.33762.7 ± 26.1
      Plasminogen (mg/dl)7.6 ± 4.47.1 ± 2.10.7998.8 ± 3.4
      Factor XI antigen (IU/ml)1.25 ± 0.251.45 ± 0.250.111
      Fibrinogen (mg/ml)2.47 ± 0.883.01 ± 0.830.219
      PAI-1 antigen (pg/ml)2077 ± 16861732 ± 10220.638
      TAFI antigen (μg/ml)5.29 ± 1.555.23 ± 2.270.949
      Pvalues compare placebo to IONIS-APO(a)Rx. End of treatment: day 85/92/99 for IONIS-APO(a)Rx and day 36 for IONIS-APO(a)-LRx; end of follow up: day 190 for IONIS-APO(a)Rx and day 113 for IONIS-APO(a)-LRx. For the continuous data, the comparisons between the two treatment arms [IONIS-APO(a)Rx treatment and placebo] in the IONIS-APO(a)Rx trial were performed using the two-sample t-test or Wilcoxon rank-sum test. For the categorical data, the comparisons between the two treatment arms in the IONIS-APO(a)Rx trial were performed using Fisher's exact test.
      Despite the highly elevated baseline Lp(a) levels and the significant reduction in Lp(a), the mean percentage change (Fig. 2) or absolute change (supplemental Fig. S1, supplemental appendix) in clot lysis times in the treatment group or placebo group showed no significant differences or any significant temporal changes. There were also no significant changes in the biomarkers of fibrinolysis and coagulation and no significant correlations between these biomarkers and clot lysis time at any time point (Table 2).
      Figure thumbnail gr2
      Fig. 2.Ex vivo clot lysis times comparing placebo to IONIS-APO(a)Rx and IONIS-APO(a)-LRx. The mean percentage change from baseline in Lp(a), clot time, and clot lysis time are shown in panels A, B, and C, respectively. ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05.
      TABLE 2.Effect of antisense therapy on coagulation/fibrinolysis biomarkers in patients treated with placebo or IONIS-APO(a)Rx
      BaselineEnd of TreatmentEnd of Follow-Up
      CharacteristicPlaceboIONIS-APO(a)RxPPlacebo (% change)IONIS-APO(a)Rx (% change)PPlacebo (% change)IONIS-APO(a)Rx (% change)P
      Lp(a) (nmol/l)362.1 ± 89.9477.3 ± 55.90.009341.9 ± 87.2 (−5.4 ± 6.9)148.0 ± 64.7 (−69.3 ± 12.2)<0.001368.3 ± 73.5 (3.2 ± 12.2)401.2 ± 50.1 (−15.6 ± 8.9)0.003
      Plasminogen (mg/dl)7.6 ± 4.47.1 ± 2.10.7997.9 ± 4.0 (14.8 ± 63.8)6.1 ± 2.1 (−5.7 ± 47.0)0.4827.1 ± 3.2 (22.1 ± 83.4)6.5 ± 3.4 (3.0 ± 63.0)0.810
      Factor XI antigen (IU/ml)1.25 ± 0.251.45 ± 0.250.1111.25 ± 0.19 (1.7 ± 13.8)1.33 ± 0.21 (−7.7 ± 7.3)0.1241.29 ± 0.25 (4.7 ± 15.5)1.36 ± 0.27 (−6.7 ± 8.9)0.102
      Fibrinogen (mg/ml)2.47 ± 0.883.01 ± 0.830.2192.59 ± 0.86 (12.7 ± 43.2)2.46 ± 0.48 (−16.5 ± 11.5)0.1042.26 ± 0.78 (−3.3 ± 30.7)2.86 ± 0.68 (−0.2 ± 30.4)0.840
      PAI-1 antigen (pg/ml)2,077 ± 1,6861,732 ± 1,0220.6383,242 ± 2,442 (65.0 ± 95.7)1,679 ± 1,544 (−5.2 ± 43.1)0.0752,543 ± 1,837 (44.7 ± 91.2)2,081 ± 1,107 (54.7 ± 87.2)0.737
      TAFI antigen (ug/ml)5.29 ± 1.555.23 ± 2.270.9495.60 ± 1.44 (8.5 ± 19.7)5.14 ± 2.66 (−2.7 ± 19.4)0.2665.70 ± 1.16 (12.4 ± 26.7)5.83 ± 2.24 (14.9 ± 23.6)0.475
      Pvalues compare placebo to IONIS-APO(a)Rx. End of treatment: day 85/92/99; end of follow-up: day 190. The comparisons between IONIS-APO(a)Rx treatment, and placebo were performed using the two-sample t-test or Wilcoxon rank-sum test.
      Substantial interindividual differences in clot lysis times were observed in the cohorts (supplemental Fig. S2, supplemental appendix). Accordingly, we also measured lysis times in a group of 16 healthy adult males with a mean ± SD age of 41.2 ± 11.7 years and range of Lp(a) concentrations [median (interquartile range): 53.5 (72.5) nmol/l]. A similar range and distribution of lysis times were observed in this cohort (supplemental Fig. S2, supplemental appendix), and no correlation between Lp(a) concentration and clot lysis time was observed (Fig. 3).
      Figure thumbnail gr3
      Fig. 3.Ex vivo clot lysis in healthy adults representing a range of Lp(a) concentrations. Baseline plasma samples for healthy adults (n = 16) enrolled in a phase 1 study of IONIS-APO(a)Rx were used. Lysis times are plotted against baseline Lp(a) concentration; no significant correlation was observed.
      In a series of control experiments, ex vivo clot lysis assays were performed using plasma from a single donor with low Lp(a) levels (12.5 nmol/l) supplemented with increasing concentrations of purified Lp(a) [single 16K apo(a) isoform] or a 17K form of recombinant apo(a). The addition of Lp(a) up to 200 nmol/l had no effect on clot lysis (Fig. 4). Similar findings were obtained using purified Lp(a) from donors with either a single 18K or double 26/28K isoforms (data not shown). In contrast, the addition of the recombinant 17K apo(a) dose-dependently increased lysis time (Fig. 4).
      Figure thumbnail gr4
      Fig. 4.Effect of purified Lp(a) or apo(a) on ex vivo plasma clot lysis. Plasma from an individual with low Lp(a) was supplemented with purified Lp(a) or 17K recombinant apo(a). Mixtures were combined with thromboplastin to induce clot formation and tPA to induce subsequent clot lysis. Clot lysis times were evaluated from measurements of turbidity over time at 37°C. Clot lysis assays were carried out in a manner similar to the patient samples in , except that tPA concentration was doubled to account for the antifibrinolytic effect of 17K. The data shown are the means ± SDs of four independent experiments. By ANOVA with Tukey's post hoc analysis, asterisks indicate significant differences between Lp(a) and apo(a). **P < 0.01 and ***P < 0.001.
      To examine the involvement of different domains of apo(a) in the inhibition of fibrinolysis, we used a series of recombinant variants (Fig. 5). Deletion of the strong lysine binding site in the KIV10 in the context of the 17K variant had no impact on its antifibrinolytic effect. Interestingly, variants smaller than 17K exhibited progressively smaller antifibrinolytic effects as they became shorter; the 6K variant, which lacks KIV1–4 and is not observed physiologically, had no antifibrinolytic effect (Fig. 5).
      Figure thumbnail gr5
      Fig. 5.Antifibrinolytic effect of recombinant apo(a) variants. A: Topology of the variants. The circle represents the mutation in KIV10 abolishing the strong lysine binding site. The bar indicates the unpaired cysteine in KIV9. KIV2 repeats are unshaded; KIV1 and KIV3–10 are lightly shaded, and KV is very lightly shaded. The protease-like domain is darkly shaded. B: Ex vivo clot lysis assays were carried as described for . The data shown are the means ± SDs of four independent experiments. By ANOVA with Dunnett's multiple comparison tests, asterisks (color-coded by variant) indicate significant differences for the respective variants compared with the absence of apo(a). *P < 0.05, **P < 0.01, and ***P < 0.001.

      DISCUSSION

      This study demonstrates that potent reductions in Lp(a) in patients with elevated Lp(a) did not result in significant changes in ex vivo clot lysis or in biomarkers of coagulation and fibrinolysis. Importantly, these patients' Lp(a) represented levels at approximately the 98–99th percentile of population levels, which are approximately >175 mg/dl or >437 nmol/l (
      • Viney N.J.
      • van Capelleveen J.C.
      • Geary R.S.
      • Xia S.
      • Tami J.A.
      • Yu R.Z.
      • Marcovina S.M.
      • Hughes S.G.
      • Graham M.J.
      • Crooke R.M.
      • et al.
      Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.
      ,
      • Varvel S.
      • McConnell J.P.
      • Tsimikas S.
      Prevalence of elevated Lp(a) mass levels and patient thresholds in 532 359 patients in the United States.
      ). Additionally, they had very small isoforms, and most had LPA SNPs associated with elevated Lp(a). Therefore, the patients in this study would be the most likely subset of patients with elevated Lp(a) to have baseline antifibrinolytic effects.
      These data are consistent with most recent studies showing no association of elevated Lp(a) with deep venous thrombosis (
      • Helgadottir A.
      • Gretarsdottir S.
      • Thorleifsson G.
      • Holm H.
      • Patel R.S.
      • Gudnason T.
      • Jones G.T.
      • van Rij A.M.
      • Eapen D.J.
      • Baas A.F.
      • et al.
      Apolipoprotein(a) genetic sequence variants associated with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism.
      ,
      • Kamstrup P.R.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Genetic evidence that lipoprotein(a) associates with atherosclerotic stenosis rather than venous thrombosis.
      ), which represents a more pure form of thrombosis than myocardial infarction or ischemic stroke where atherosclerosis is concomitantly present. Lp(a) may not exert a major effect on thrombosis in vivo, and its association with myocardial infarction may be attributable to effects on inflammation, oxidation, and atherosclerosis as primary mechanisms (
      • Tsimikas S.
      • Fazio S.
      • Ferdinand K.C.
      • Ginsberg H.N.
      • Koschinsky M.L.
      • Marcovina S.M.
      • Moriarty P.M.
      • Rader D.J.
      • Remaley A.T.
      • Reyes-Soffer G.
      • et al.
      NHLBI Working Group recommendations to reduce lipoprotein(a)-mediated risk of cardiovascular disease and aortic stenosis.
      ).
      It is notable that a substantial interindividual variability is observed in clot lysis time, both in our subjects with very high Lp(a) levels as well as a sample of healthy adults representing a broad range of baseline Lp(a) concentrations. In agreement with the lack of impact of Lp(a) lowering on lysis times, there was no correlation between baseline Lp(a) levels and clot lysis time.
      Allowing for the fact that the current ex vivo assays may not ideally reflect in vivo conditions, it is possible that prothrombotic effects of Lp(a) in vivo are present beyond the inhibition of fibrinolysis and that are not captured by this assay; some previously documented examples include the promotion of platelet activation (
      • Rand M.L.
      • Sangrar W.
      • Hancock M.A.
      • Taylor D.M.
      • Marcovina S.M.
      • Packham M.A.
      • Koschinsky M.L.
      Apolipoprotein(a) enhances platelet responses to the thrombin receptor-activating peptide SFLLRN.
      ) and inhibition of TFPI (
      • Caplice N.M.
      • Panetta C.
      • Peterson T.E.
      • Kleppe L.S.
      • Mueske C.S.
      • Kostner G.M.
      • Broze Jr., G.J.
      • Simari R.D.
      Lipoprotein (a) binds and inactivates tissue factor pathway inhibitor: a novel link between lipoproteins and thrombosis.
      ). In addition, contradictory data have been reported on the relationship between rs3798220 carrier status and clot lysis times ex vivo (
      • Rowland C.M.
      • Pullinger C.R.
      • Luke M.M.
      • Shiffman D.
      • Green L.
      • Movsesyan I.
      • Devlin J.J.
      • Malloy M.J.
      • Kane J.P.
      • Undas A.
      Lipoprotein (a), LPA Ile4399Met, and fibrin clot properties.
      ,
      • Chandler P.D.
      • Song Y.
      • Lin J.
      • Zhang S.
      • Sesso H.D.
      • Mora S.
      • Giovannucci E.L.
      • Rexrode K.E.
      • Moorthy M.V.
      • Li C.
      • et al.
      Lipid biomarkers and long-term risk of cancer in the Women's Health Study.
      ). In general, the stoichiometry does not favor in vivo inhibition of plasminogen activity by Lp(a) because the plasma levels of plasminogen (5–20 mg/dl or 543–2,174 nmol/l) are usually in excess to Lp(a) even with the highest levels of Lp(a) at the 99th percentile of levels (437 nmol/l). However, antifibrinolytic effects of Lp(a) may be manifest in local microenvironments in which excessive accumulation of Lp(a) may prevent optimal plasminogen activation to plasmin to effect fibrinolysis in preexisting thrombi. Furthermore, Lp(a) may be synergistic with other prothrombotic risk factors and may not have a significant effect on its own (
      • Foody J.M.
      • Milberg J.A.
      • Robinson K.
      • Pearce G.L.
      • Jacobsen D.W.
      • Sprecher D.L.
      Homocysteine and lipoprotein(a) interact to increase CAD risk in young men and women.
      ).
      The lack of effect of purified Lp(a) on clot lysis is surprising in light of an extensive body of evidence that apo(a) itself inhibits fibrinolysis and plasminogen activation (
      • Boffa M.B.
      • Koschinsky M.L.
      Lipoprotein (a): truly a direct prothrombotic factor in cardiovascular disease?.
      ). Only one study utilized our approach of adding Lp(a) to plasma and measuring tPA-mediated clot lysis, and the authors found that ∼45 nmol/l Lp(a) reduced the extent of clot lysis by at most 25%, although the method used for preparing the clots and monitoring clot lysis was different from the current method (
      • Loscalzo J.
      • Weinfeld M.
      • Fless G.M.
      • Scanu A.M.
      Lipoprotein(a), fibrin binding, and plasminogen activation.
      ). Several studies have detected correlations between Lp(a) levels and the inhibition of plasma clot lysis in vitro with Lp(a)-lowering hormone replacement therapy (
      • Falcó C.
      • Tormo G.
      • Estelles A.
      • Espana F.
      • Tormo E.
      • Gilabert J.
      • Velasco J.A.
      • Aznar J.
      Fibrinolysis and lipoprotein(a) in women with coronary artery disease. Influence of hormone replacement therapy.
      ) or in the setting of disorders such as residual vein obstruction and cerebral venous sinus thrombosis (
      • Undas A.
      • Ciesla-Dul M.
      • Drazkiewicz T.
      • Sadowski J.
      Altered fibrin clot properties are associated with residual vein obstruction: effects of lipoprotein(a) and apolipoprotein(a) isoform.
      ), or myocardial infarction (
      • Undas A.
      • Plicner D.
      • Stepien E.
      • Drwila R.
      • Sadowski J.
      Altered fibrin clot structure in patients with advanced coronary artery disease: a role of C-reactive protein, lipoprotein(a) and homocysteine.
      ). In another study, it was found that patients with type 2 diabetes mellitus had longer clot lysis times than healthy control subjects, although no difference in lysis time by quartile of Lp(a) levels was found in either group (
      • Månsson M.
      • Kalies I.
      • Bergstrom G.
      • Schmidt C.
      • Legnehed A.
      • Hulten L.M.
      • Amrot-Fors L.
      • Gustafsson D.
      • Knecht W.
      Lp(a) is not associated with diabetes but affects fibrinolysis and clot structure ex vivo.
      ). Our study has an advantage over these in that we used individual subjects receiving either placebo or IONIS-APO(a)Rx as their own control, minimizing selection bias and other confounders.
      In contrast to Lp(a), the human apo(a) isoform 17K had a clear antifibrinolytic effect in our assay, in agreement with previous findings from both in vitro and in animal models (
      • Palabrica T.M.
      • Liu A.C.
      • Aronovitz M.J.
      • Furie B.
      • Lawn R.M.
      • Furie B.C.
      Antifibrinolytic activity of apolipoprotein(a) in vivo: human apolipoprotein(a) transgenic mice are resistant to tissue plasminogen activator-mediated thrombolysis.
      ,
      • Sangrar W.
      • Bajzar L.
      • Nesheim M.E.
      • Koschinsky M.L.
      Antifibrinolytic effect of recombinant apolipoprotein(a) in vitro is primarily due to attenuation of tPA-mediated Glu-plasminogen activation.
      ,
      • Biemond B.J.
      • Friederich P.W.
      • Koschinsky M.L.
      • Levi M.
      • Sangrar W.
      • Xia J.
      • Buller H.R.
      • ten Cate J.W.
      Apolipoprotein(a) attenuates endogenous fibrinolysis in the rabbit jugular vein thrombosis model in vivo.
      ). These data suggest that, compared with Lp(a), free apo(a) is a more potent antifibrinolytic agent; this may explain some of the findings in the literature with such constructs (
      • Boffa M.B.
      • Koschinsky M.L.
      Lipoprotein (a): truly a direct prothrombotic factor in cardiovascular disease?.
      ). However, the clinical significance of this is not clear, as very little (<5%) free apo(a) exists in human plasma of untreated patients, except perhaps in renal failure (
      • Edelstein C.
      • Pfaffinger D.
      • Hinman J.
      • Miller E.
      • Lipkind G.
      • Tsimikas S.
      • Bergmark C.
      • Getz G.S.
      • Witztum J.L.
      • Scanu A.M.
      Lysine-phosphatidylcholine adducts in kringle V impart unique immunological and potential pro-inflammatory properties to human apolipoprotein(a).
      ,
      • Arai K.
      • Orsoni A.
      • Mallat Z.
      • Tedgui A.
      • Witztum J.L.
      • Bruckert E.
      • Tselepis A.D.
      • Chapman M.J.
      • Tsimikas S.
      Acute impact of apheresis on oxidized phospholipids in patients with familial hypercholesterolemia.
      ,
      • Mooser V.
      • Marcovina S.M.
      • Wang J.
      • Hobbs H.H.
      High plasma levels of apo(a) fragments in Caucasians and African-Americans with end-stage renal disease: implications for plasma Lp(a) assay.
      ). In the current era of achieving very low plasma apoB-100 levels (
      • Giugliano R.P.
      • Pedersen T.R.
      • Park J.G.
      • De Ferrari G.M.
      • Gaciong Z.A.
      • Ceska R.
      • Toth K.
      • Gouni-Berthold I.
      • Lopez-Miranda J.
      • Schiele F.
      • et al.
      Clinical efficacy and safety of achieving very low LDL-cholesterol concentrations with the PCSK9 inhibitor evolocumab: a prespecified secondary analysis of the FOURIER trial.
      ), it is conceivable that inadequate amounts of apoB-100 will be available for Lp(a) particles to be generated and that increased amounts of free apo(a) may circulate, a paradigm shown previously in Lp(a)-transgenic mice (
      • Merki E.
      • Graham M.
      • Taleb A.
      • Leibundgut G.
      • Yang X.
      • Miller E.R.
      • Fu W.
      • Mullick A.E.
      • Lee R.
      • Willeit P.
      • et al.
      Antisense oligonucleotide lowers plasma levels of apolipoprotein (a) and lipoprotein (a) in transgenic mice.
      ) and that we have postulated may occur in patients (
      • Yeang C.
      • Witztum J.L.
      • Tsimikas S.
      ‘LDL-C’ = LDL-C + Lp(a)-C: implications of achieved ultra-low LDL-C levels in the proprotein convertase subtilisin/kexin type 9 era of potent LDL-C lowering.
      ).
      One potential reason why apo(a) may not inhibit fibrinolysis in the context of the Lp(a) particle is that the kringle domains involved are blocked by association with apoB-100. We do not believe that this is likely because the recombinant apo(a) would rapidly associate with the excess LDL present in the plasma samples. We also performed experiments in which we added purified LDL up to 400 nmol/l (particle concentration) to the assays, but this did not blunt the antifibrinolytic effect of 17K (data not shown).
      Our experiments with recombinant variants of apo(a) also yielded some unexpected findings. First, the strong lysine binding site in KIV10 [the kringle that also harbors the covalently bound oxidized phospholipid in apo(a) (
      • Leibundgut G.
      • Scipione C.
      • Yin H.
      • Schneider M.
      • Boffa M.B.
      • Green S.
      • Yang X.
      • Dennis E.A.
      • Witztum J.L.
      • Koschinsky M.L.
      • et al.
      Determinants of binding of oxidized phospholipids on apolipoprotein(a) and lipoprotein(a).
      )] is dispensable for inhibiting fibrinolysis. This differs from our previous findings that ablation of this site lowers the ability of apo(a) to inhibit tPA-mediated plasminogen activation and to attenuate plasmin-mediated conversion of Glu-plasminogen to Lys-plasminogen (
      • Feric N.T.
      • Boffa M.B.
      • Johnston S.M.
      • Koschinsky M.L.
      Apolipoprotein(a) inhibits the conversion of Glu-plasminogen to Lys-plasminogen: a novel mechanism for lipoprotein(a)-mediated inhibition of plasminogen activation.
      ,
      • Hancock M.A.
      • Boffa M.B.
      • Marcovina S.M.
      • Nesheim M.E.
      • Koschinsky M.L.
      Inhibition of plasminogen activation by lipoprotein(a): critical domains in apolipoprotein(a) and mechanism of inhibition on fibrin and degraded fibrin surfaces.
      ). We also found that smaller apo(a) variants, including rare but clinically observable 14K and 12K variants, display a drastically reduced antifibrinolytic potency. The 6K variant, which potently inhibits tPA-mediated plasminogen activation and Lys-plasminogen formation, had no antifibrinolytic effect at all (
      • Feric N.T.
      • Boffa M.B.
      • Johnston S.M.
      • Koschinsky M.L.
      Apolipoprotein(a) inhibits the conversion of Glu-plasminogen to Lys-plasminogen: a novel mechanism for lipoprotein(a)-mediated inhibition of plasminogen activation.
      ,
      • Hancock M.A.
      • Boffa M.B.
      • Marcovina S.M.
      • Nesheim M.E.
      • Koschinsky M.L.
      Inhibition of plasminogen activation by lipoprotein(a): critical domains in apolipoprotein(a) and mechanism of inhibition on fibrin and degraded fibrin surfaces.
      ,
      • Fortunato J.E.
      • Bassiouny H.S.
      • Song R.H.
      • Kocharian H.
      • Glagov S.
      • Edelstein C.
      • Scanu A.M.
      Apolipoprotein (a) fragments in relation to human carotid plaque instability.
      ). These results indicate that fragments of apo(a) generated by proteolysis of Lp(a) in vivo would not generate species capable of inhibiting fibrinolysis.
      Limitations of this study include the small study size, and thus these data need to be confirmed in larger studies. Furthermore, ex vivo clot lysis assays may not necessarily reflect in vivo properties of Lp(a) on fibrinolysis. Finally, the variation in the clot lysis times for a given patient observed at the different time points may have obscured any effect of Lp(a) lowering.
      In conclusion, in patients with highly elevated Lp(a), we find no evidence that a very potent reduction in Lp(a) affects ex vivo fibrinolysis. These observations suggest the main pathophysiological contribution of Lp(a) to CVD and aortic stenosis may be through its proatherogenic and proinflammatory components, such as oxidized phospholipids (
      • Boffa M.B.
      • Koschinsky M.L.
      Oxidized phospholipids as a unifying theory for lipoprotein(a) and cardiovascular disease.
      ). Further investigation is required using larger cohorts and additional methodologies to confirm these findings.

      Acknowledgments

      The authors thank Santica Marcovina for providing purified Lp(a) of different isoform sizes.

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