Lipoprotein (a) blood levels in unstable angina pectoris, acute myocardial infarction, and after thrombolytic therapy

Lipoprotein (a) Blood l+vels in Unstable Angina Pectoris, Acute Myocardial Infarction, and After Thrombolytic Therapy Shiqiang Qiu, MD, Pierre Thkroux, MD, Jacques Genest, Jr., MD, B. Charles Solymoss, MD, Danielle Robitaille, MD, and Michel Marcil, BSC

Lipoprotein (a) [Lp(a)] appears to be involved in atherogenesis and in vitro studies have suggested that it may interfere with thrombolysis. In this study, Lp(a) serum levels were determined by radioimmunoassay in 124 patients with ischemic heart disease. Of these, 47 had acute myocardial infarction, 13 had unstable angina, and 64 were age-matched patients with stable angina. Of the 60 patients with acute coronary artery disease, 34 received thrombolysis and 26 did not. In addition to Lp(a), serum plasminogen, CY~antiplasmin, fibrinogen, and D-dimer (cross-linked fibrin degradation products) levels were measured. These tests were repeated after 6 hours in patients with myocardial infarction and unstable angina. No significant difference was found for admission Lp(a) levels among patients with myocardial infarction (0.324 f 0.047 g/liter), unstable angina (0.435 f 0.123 g/liter) and stable angina (0.431 f 0.023 g/liter), between patients with myocardial infarction with or without thrombolytic treatment, nor between late and early measurements in patients with unstable angina and acute myocardial infarction. Plasminogen, a2 antiplasmin and fibrinogen values decreased significantly after thrombolytic treatment. The size of this decrease correlated positively with higher Lp(a) blood levels (p <0.05). Patients with &p(a) >0.25 g/liter had a 66% decrease in fibrinogen and a 53% decrease in antiplasmin, compared with 35 and 32%, respectively, in patients with Lp(a) level 10.25 g/liter (p <0.05). Plasminogen levels revealed a similar trend, with a 61% decrease for the higher values and a 45% decrease for the lower values. These data indicate that serum Lp(a) levels are not increased in acute myocardial infarction and unstable angina, and are not influenced by thromFrom the Montreal Heart Institute, Montreal, Quebec, Canada. Manuscript received January 4, 1990; revised manuscript received and accepted January 24, 199 1. Address for reprints: Pierre Theroux, MD, Montreal Heart Institute, 5000 East, Belanger Street, Montreal, Quebec HIT lC8, Canada.

bolytic found nolytic nostic

treatment. In addition, no evidence was that higher levels reduced systemic fibriactivity, as assessed by the usual diagtests. (Am J Cardiol 1991;67:1175-1179)

r&able angina and myocardial infarction are acute manifestations of coronary artery disease, related to plaque rupture,’ platelet aggregation, thrombin activation2 and formation of an occlusive intracoronary thrombus.3 These processes also stimulate endogenous fibrinolysis; the balance between procoagulants and lytic forces could be 1 of the determinants of ultimate clinical outcome. Pharmacologic fibrinolysis has now become routine treatment of patients with acute myocardial infarction in an effort to restore coronary blood flow promptly.4 Lipoprotein (a) [Lp(a)] is composed of 1 low-density lipoprotein particle to which is covalently bound, to the apolipoprotein (apo) B moiety, > 1 molecule of apo(a).5,6 Ape(a) is a glycosylated protein that shares considerable homology with plasminogen.7 The gene for ape(a) is adjacent to that of plasminogen on chromosome 6 (6827) and is composed of multiple repeats of kringle IV-like domains.* Although the role of Lp(a) is poorly understood, it interferes with thrombolysis by decreasing the rate of plasminogen activation by streptokinase or tissue plasminogen activator by binding to fibrin and by competing with plasminogen for its receptor on vascular endothelial cells.9-13 In this study, we assessedserum levels of Lp(a) during the acute process of unstable angina and acute myocardial infarction and examined the interaction of these levels with markers of fibrinolytic activity during thrombolysis.

U

METHODS Study patients: The study group comprised 124 hospitalized patients, 60 with an acute manifestation of coronary artery disease and 64 with stable angina. Patients with acute disease were a consecutive series of patients admitted for 2 1 episode of chest pain ~30 minutes in duration, not relieved by sublingual nitroLP(A)

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glycerin, and accompanied by an ST-segment shift or T-wave inversion on the 12-lead electrocardiogram. Serial creatine kinase and MB-isoenzyme blood levels were obtained, and acute myocardial infarction was diagnosed when total creatine kinase values doubled with the MB fraction present; unstable angina was diagnosed when no such enzyme elevation was observed. Myocardial infarction occurred in 47 patients and unstable angina in 13. Patients were further divided into a group of 34 patients who received thrombolytic therapy and a group of 26 who did not. The thrombolytic agents used were streptokinase in 29 patients at a dose of 1.5 million U administered intravenously during 30 to 45 minutes or single-chain, recombinant tissue-type plasminogen activator (Activase@, Genentech Corporation) in 5 patients at a total dose of 100 mg administered over 4 hours with the step-down infusion protocol recommended by the manufacturer. All patients who received thrombolysis had ST-segment elevation on their initial electrocardiogram; one-third of those who did not also had ST elevation. The patients with stable angina were selected by computer in a random order from a pool of 900 patients hospitalized for elective cardiac catheterization requested by the treating physician. Criteria for their selection included the presence of angiographically documented coronary artery disease with percent luminal diameter reduction of 250% in the right or left coronary artery. They were further selected by computer to match the age and sex of the group of patients with acute coronary artery disease. Blood sampling and analyses: Blood analyses performed included total creatine kinase and its MB fraction, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, Lp(a), plasminogen, fibrinogen, a~ antiplasmin and D-dimers levels. In patients with unstable coronary artery disease, cardiac enzymes were determined at admission, every 2 hours for 6 hours and every 4 hours for the following 24 hours. Lp(a) values were obtained at admission before the administration of any specific treatment for acute myocardial infarction or unstable angina and 6 hours later. Fibrinolytic activity measured by levels of plasminogen, fibrinogen, antiplasmin and D-dimers were similarly assessed at admission and 6 hours later. The lipid profile, excluding Lp(a), was obtained in the fasting state between 8 to 18 hours after admission. In patients with stable angina, the lipid measurements were performed the morning after admission, after a 12-hour fasting period and before angiography. Creatine kinase blood levels were measured with optimized reagents from Boehringer-Mannheim and its

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MB fractions by immunoinhibition reagent from Boehringer-Mannheim, verified if necessary by agarose electrophoresis. Blood samples for lipid determination were obtained in tubes with no anticoagulant, and for coagulation factor determination in tubes with buffered sodium citrate. These were immediately centrifuged and the plasma stored at -7O“C for subsequent batch analyses. Total blood cholesterol, HDL cholesterol and triglyceride levels were measured by enzymatic techniques with standards and reagents from Boehringer-Mannheim. The precipitating agent used for HDL cholesterol was low-molecular-weight dextran sulfate with magnesium chloride. LDL cholesterol levels were calculated using the Friedewald equation. l4 Lp(a) blood levels were analyzed by radioimmunoassay with antisera standards and control materials from Pharmacia Diagnostic AB (Uppsala, Sweden). Cross reactivity with plasminogen and apo B is absent up to concentrations of 8.5 g/liter of plasminogen and 7.0 g/liter of apo B in this assay.15 Serum fibrinogen, plasminogen and cy2antiplasmin were measured by the Automated Coagulation Laboratory (Fisher Scientific Ltd, Milan, Italy). Thromboplastin from Baxter Co. Ltd (Miami, Florida) was used for the fibrinogen assay. The plasminogen assay was performed with the samples incubated in an excess of streptokinase and the plasmin-like activity detected on a synthetic chromogenic substrate. Reagents for plasminogen and a!2 antiplasmin were from Instrumentation Laboratory SPA (Milan, Italy). The samples were incubated with an excess of plasmin in biologic conditions minimizing the influence of other plasmin inhibitors, and residual plasmin activity was measured on the synthetic chromogenic substrate. Determination of Ddimers was made semiquantitatively with the Latex slide test (Ortho Dimer test, Ortho Diagnostic Systems, Beerse, Belgium). Statistical analysis: Data were analyzed using the BMDP statistical software package (Los Angeles, California). Baseline characteristics of the various study groups were compared using the t test for continuous data and chi-square analysis for discrete data after having assessed any significant departure of the data from the mean. Paired t tests were used when comparing changes in values within the same subjects. Because Lp(a) blood levels followed a non-normal distribution, they were analyzed using the nonparametric MannWhitney analysis for nonmatched comparisons of 2 populations. An Lp(a) cut-off value of 0.25 g/liter served to discriminate normal or abnormally high values. The semiquantitative data of D-dimers levels were analyzed by a significant test of rate of 2 samples. The level of a p value <0.05 was considered statistically sig-

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TABLE I Demographic Characteristics the Various Study Groups*

and Lipid Profiles

of

Groups

Myocardial

Unstable

Study Groups

Infarction

Angina

Stable Angina

No. of pts. Male/female

47

Age Ws)

55*7 5.06f0.25

13 7/6 64~1~4 5.65zk0.47

64 al/20 52+2 6.09iO.14

3.56zkO.29

3.58+0.37

3.95f0.12

0.98f0.04

0.94f0.12

1.03-+0.03

1.50f0.12

2.51 f 0.65

2.40f0.19

Total cholesterol (mmol/liter) LDL cholesterol (mmol/liter) HDL cholesterol (mmol/liter) Triglycerides (mmol/liter)

39/S

TABLE II Lipoprotein

* No statistically significant differences exist between any of the study groups. HDL = highdensity lipoprotein; LDL = low-density lipoprotein.

nificant. Values are expressed as mean f standard error of the mean. RESULTS

Mean age of the study population was 54 f 12 years, and 73% were men. Clinical characteristics and lipid profiles of the various study groups are listed in Table I. These were evenly distributed; a trend to higher total blood cholesterol was present in patients with stable angina. The Lp(a) levels in the 3 groups as well as in patients with myocardial infarction who did or did not receive thrombolytic therapy are listed in Table II, which also lists the levels observed before and 6 hours after administration of streptokinase or recombinant tissue-type plasminogen activator. The mean value of the lipoprotein in patients with acute coronary syndrome (unstable angina and myocardial infarction) was 0.350 f 0.046 g/liter and in patients with stable angina 0.431 f 0.023 g/liter (p = 0.3). Patients with acute disease who did or did not undergo thrombolysis also had similar baseline levels-O.356 f 0.056 and 0.342 f 0.073 g/liter, respectively. The use of a thrombolytic agent (streptokinase or recombinant tissue-type plasminogen activator) did not modify Lp(a) levels assessed 6 hours after initiation of treatment: 0.356 f 0.056 compared with 0.342 f 0.073 g/liter after 6 hours. However, plasminogen levels decreased from 1.169 f 0.055 to 0.504 f 0.061 g/liter (p 0.25 g/ liter) Lp(a) levels. After induced tibrinolysis, normal Lp(a) levels were associated with mean plasminogen

and Before

(a) Blood Levels and After Fibrinolysis

Stable angina Acute coronary disease Myocardial infarction Unstable angina Thrombolysis No thrombolysis Before thrombolysis After thrombolysis

in the Various

Study

No.

Lp(a)*

64 60 47 13 34 26 34

0.431f0.023 0.35OiO.046 0.324f0.047 0.431f0.123 0.356f0.056 0.342f0.073 0.356f0.056 0.326f0.067

(g/liter)

* Mean f standard error of the mean; no statistically significant differences existed between any of the subgroups.

levels of 0.595 f 0.105 g/liter, versus 0.398 f 0.035 with high values (p = O.l), QI:! antiplasmin levels of 0.659 f 0.092 IU/liter versus 0.394 f 0.029 (p = 0.01) and fibrinogen levels of 2.181 f 0.344 g/liter versus 1.370 f 0.212 (p = 0.05) (Figure 1). The percent decreases were 45 f 12 versus 61 f 5% for plasminogen, 35 f 11 versus 53 f 13% for cy2antiplasmin and 32 f 13 versus 65 f 5% for fibrinogen. D-dimer levels increased to the detectable range in 8 of the 29 patients who received thrombolytic treatment and in only 1 of the 22 patients who did not (p
This study documents similar Lp(a) levels in patients in the acute phase of unstable angina and myocardial infarction and in patients with stable coronary artery disease. Furthermore, it shows that higher levels are not associated with a significant inhibition of the markers of fibrinolysis. Rather, high levels were associated with a significantly greater decrease in 012antiplasmin and in fibrinogen levels. The association between Lp(a) levels and atherosclerosis has been documented previously. 16-20 Higher levels are associated with more severe coronary artery disease16 and premature manifestation of the disease.17J8 Familial aggregation studies have shown a 2.5% higher risk, with levels above 0.25 g/liter.19 The

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TABLE III Clinical Characteristics

and Lipid Profiles with Normal or High Lipoprotein (a) Blood with Acute lschemic Syndromes

Associated in Patients

Lp(a)

No. of pts. Lp(a) (g/liters) Age (v-9 Male/female Smokers Total cholesterol (mmol/liter) LDL cholesterql (mmol/liter) HDL cholesterol (mmol/liter) Triglyceridds (mmol/liter) Thrombolytic treatment With rt-PA With streptokinase

Levels

Blood Levels S0.25g/liter

>0.25

33 0.107 f 0.015 55f2 27/6 19 5.29 f 0.27

27 0.647 f 0.064 59f2 19/S 16 5.17f0.21

g/liter

3.65 zt 0.32

3.39 f 0.20

0.98 f 0.05

1.02 f 0.05

1.84k0.23

1.58f0.14 17 1

18 4 14

16

Lp(a) = lipoprotein (a); rt-PA = recombinant other abbreviations as in Table I.

tissue-type plasminogen activator;

increase in risk is independent of other predictorsi6-18; combined with high LDL cholesterol values, this increase is in the range of 5%.16,17 The atherogenecity of ape(a) can be related to its strong binding to the glycosoaminoglycans of the arterial wa1121 and to increased cholesterol ester accumulation in macrophages. 22 The lipid content of Lp(a) is

Plasminogen

cd-

carried by apo B-100. This apo B-100 molecule, unique among lipoproteins, is linked to a high-molecular-mass glycoprotein, ape(a). Ape(a) contains the cation domain of serine protease esterase, which is nearly identical to the domain of plasminogen responsible for its linkage to the receptor. It lacks, however, the protein esterase arginine site responsible for the cleavage of fibrin; this site is replaced by a serine site that possesses no action on fibrin. Experimental studies have indeed documented that Lp( a) decreases streptokinase-mediated lysis of fibrin clot9 by competing directly with the plasminogen receptors.10-12 A plasma concentration of 0.25 to 0.40 g/liter could occupy 20% of the receptors,12 resulting in a corresponding reduction in fibrinolytic activity. In this study, we investigated whether these observations could be extended to the acute phase of unstable angina and myocardial infarction. No clear association could be demonstrated: Mean levels of Lp(a) were similar in all study groups. It is noted, however, that all patients had documented coronary artery disease. Total cholesterol and LDL cholesterol values were slightly less in patients with unstable angina or acute myocardial infarction, in accordance with previous observations of decreased levels during acute ischemic states.23 Transient changes in serum Lp(a) have also been reported in acute myocardial infarction, implying that the lipoprotein may respond like an acute phase

Fibrinogen

a,-Antiplasmin

,

cl/L

1.2 -

0.9

5-

4-

-

3-

I_;

0.6

\

.Oi

ns

2 ;h

0.3

0.3

p < .05

1

1

0.0 -

0.0 Before

fibrinolysis

After

O-

Before

fibrinolysis

After

fibrinolysis Lp(a)

serum

level

fibrinolysis + -R

Before fibrinolysis

$25 g/L > 25glL

FIGURE 1. Fibrinoiytic activity measured before and 6 hours after administration of the thromboiytic plasminogen, CQ antiplasmin and in fibrinogen is greater in patients with high lipoprotein (a) [l.p(a)] with lower values, suggesting greater activation of fibriaoiysis in these patients.

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After fibrinolysis

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agent. The decrease in blood levels than in patients

reactant to inflammatory processes.24 However, maximal levels in that study were reached after 5 to 10 days, showing a slow-phase reaction; in our study, levels were measured after only 6 hours. Although the acute thrombotic event was fiat accompanied by an acute elevation of Lp(a), compared with patients with stable angina, an important role of the apolipoprotein at the local level cannot be excluded. The triggers for thrombotic events appear local and related to plaque characteristics and local blood flow rheology. Thus, local thrombogenic forces may overwhelm local fibrinolysis, resulting in clot formation. In this study, thrombotic activity was not measured and higher thrombotic activity could be an explanation for t,he enhanced fibrinolytic activity observed with higher Lp(a) levels. Thus, it could be hypothesized that the accentuated fibrinolysis and fibrinogenolysis present with Lp(a) levels >0.25 g/liter could paradoxically reflect reduced local fibrinolytic activity. In addition to the complexity of extrapolating systemic observations to a local problem, the interpretation of our results is further complicated by the size polymorphism of ape(a). The apolipoprotein presents in multiple isoforms, varying in molecular weight from 300 to 700 KD. To date, 11 isoforms have been described25 and the size heterogeneity appears to be due to varying numbers of kringle IV-like domains in the molecule. This size polymorphism influences total levels of Lp(a).8 In this study, the isoforms of Lp(a) were not measured. REFERENCES 1. Davies infarction,

MJ, Thomas AC. Plaque fissuring-the sudden ischemii: death, and crescendo

53:363-373. 2. ThBroux P, L&our

cause of acute myocardial angina. Er Heart J 1985;

JG, L6gerGauthier C, De Lam J. Fibrinopeptide A and platelet factor levels in unstable angina. CircuIation 1987;75:156-162. 3. DeWood MA, Spores J, Notske R, Mouser LT, Burroughs R, Golden MS, Lang HT. Prevalence of total coronary artery occlusion during the early hours of transmural myocardial infarction. N Engl .Z Med 1980;303:897-902. 4. Gruppo Italian0 per lo Studio Della Streptwhinasi Nell’Infarto Miocardico (GISSI). Effectiveness of intravenous thrombolytic treatment in acute myocardial infarction. Lancet 1986;1:397-401. 5. Berg K. A new serum type system in man: the Lp(a) system. Acta Path&

Microbid Stand 1963;59:369-382. 6. Utermann G, Menzel HJ, Kraft HG, Duba HC, Kemmler HG, Seitz C. Lp(a) glycoprotein phenotypes. Inheritance and relation to Lp(a) lipoprotein concentration in plasma. J Clin Invest 1987;80:458-465. 7. McLean JW, Tomlinson JE, Kuang WJ. cDNA sequence of human apolipoprotein (a) is homologous to plasminogen. Nature 1987;330:132-137. 6. Utermann G. The mysteries of lipoprotein (a). Science 1989;246:904-910. 9. Karadi I, Kostner GM, Gries A, Nimpf J, Romics L, Malle E. Lipoprotein (a) and plasminogen are immunochemically related. Biochim Biophys Acta 1988;

960191-97. 10. Edelberg JM, Gonzalez-Gronow M, Pizza SV. Lipoprotein a inhibits streptokinase-mediated activation of human plasminogen. Biochemistry 1989;28:2370-

2374. 11. Gonzalez-Gronow

M, Edelberg JM, Pizza SV. Further characterization of the cellular plasminogen binding site: evidence that plasminogen 2 and lipoprotein a compete for the same site. Biochemistry 1989;28:2374-2377. 12. Miles LA, Fless GM, Levin EG, Scanu AM, Plow Ei‘. A potential basis for the thrombotic risks assqciated with lipoprotein (a). Nature 1989;339:301-303. 13. Hajjar KA, Gavish D, Breslow JL, Nachman RL. Lipoprotein (a) mcdulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis.

Nature 1989;339:303-305. 14. Fricdewald WT, Levy RI, Frederickson

DS. Estimation of low-density lip+ protein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972;1&499-508. 15. Holmlund E, Frimodig C, Jansson G, Osterberg M. Benefit and practicability of a commercial solid phase monoclon~l ape(a) RIA assay. Uppsala, Sweden: Pharmacia Diagnostics AB, 1989. 16. Armstrong VW, Cremer P, Eberle E, Manke A, Schulze F, Wieland H, Krewer H, Seidel D. The association between serum Lp(a) concentrations and angiographically assessed coronary atherosclerosis-dependence on serum LDL levels. Atherosclerosis 1986;62:249-257. 17. Rhoads GC, Dahlen G, Berg K, Morton NE, Dannenberg AL. Lp(a) lipoprotein as a risk factor for myocardial infarction. .ZAMA 1986;256:2540-2544. 16. gandkamp M, Funke H, Schulte H, Khdler E, Assmann G. Lipoprotein (a) is an independent risk factor for myocardial infarction at a young age. Chin Chem 1990;36:20-23. 19. Hoefler G, tiarnoncourt F, Paschke E, Mirtl W, Pfeiffer KH, Kostner GN. Lipoprotein Lp(a). A risk factor for myocardial infarction. Arteriosclerosis 1988;8:398-401. 20. Murai A, Miyahava T, Fujimoto N, Matsuda M, Kameyana M. Lp(a) lipoprotein as a risk factor for coronary heart disease and cerebral infarction. Atherosclerosis 1986;59:199-206. 21. Dahlen G, Ericson C, Berg K. In vitro studies on the interaction of isolated Lp(a) lipoprotein and other serum lipoproteins with glycosoaminoglycans. CZin Genet 1978;14:36-42. 22. Krempler F, Kostner GM, Roscher A, Bolzano K, Sandhofer F. The interao tion of human ape B-containing lipoproteins with mouse peritoneal macrophages: a comparison of Lp(a) with LDL. d Lipid Res 1984;25:283-287. 23. Ryder REJ, Hayes TM, kulligaa IP, Kingswood JC, Williams S, Owens DR. How soan after myoeardial infarction should plasma lipid values be assessed? Br Med J 1984;289:1638-1640. 24. Maeda S, Abe A, Seishima M, Makino K, Noma A, Kawade M. Transient changes of serum lipoprotein (a) as an acute phase protein. Atherosclerosis 1989;78:145-150. 25. Gavish D, Azrolan N, Breslow JL. Plasma Lp(a) concentrations is inversely correlated with the ratio of kringle IV/kringle V encoding domains in the ape(a) gene. J Chin Znuest 1989;84:2021&2027.

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