THROMBOSIS RESEARCH 63; 407-418,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.

FIBRINOLYTIC PARAMETERS AND LIPOPROTEIN (a) LEVELS IN PLASMA OF PATIENTS WITH CORONARY ARTERY DISEASE

L.J. Garcia Frade, J.J. Alvarez* , I. Rayo"". M.C. Torrado, M.A. Lasuncion", A. Garcia Avello, A. Hernandez, E. Marin"". Departments of Haematology, Biochemistry* and Cardiology"*. Hospital Ramon y Cajal, Alcala University, Madrid, Spain.

(Received 8.2.1991; accepted in revised form 3.6.1991 by Editor S. Moncada)

ABSTRACT Fibrinolysis and lipid disturbances have been considered as independent risk factors for coronary artery disease. Besides this, lipoprotein(a), which is characterized by plasminogen may interfere with the its homology with eventual fibrinolytic function. To evaluate the correlation between fibrinolytic parameters, lipoprotein (a) and other risk factors, 46 patients with coronary and 12 artery disease (34 with chronic angina pectoris Increased with myocardial infarction) were studied. 4.2 ng/ml) basal values of t-PA antigen (8.2 and 6.6 vs. but decreased response after stimulous (2.2 and I.8 vs. 3.8 ng/ml) and increased levels of lipoprotein(a) (24.7 relevant and 35.9 vs. 10.5 mg/dl) were the most differences between coronary artery disease patients and controls. No correlation between lipoprotein(a) and fibrinolytic parameters was found. Therefore plasma concentration of the main plasma fibrinolytic parameters and lipoprotein(a) seem to be unrelated though the relevance of this interaction at a local level needs to be studied.

INTRODUCTION Chronic stable angina and myocardial infarction are two common manifestations of coronary artery disease (CAD). Several factors such as arterial hypertension, diabetes, obesity, Keywords: Coronary lipoprotein(a).

artery

disease,

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hyperlipemia. physical inactivity and tobacco have been considered as risks for coronary artery disease (1). These factors have abnormalities in been also associated to fibrinolysis (2, 3. 4, 5). The fibrinolytic system seems to be implicated in the development of thrombosis and atherosclerosis. Also fibrinolytic function has been related to mortality from cardiovascular disease as was shown in the Northwick Park Heart Study (6). where fibrinolytic activity tended to be lower in subjects who later died from cardiovascular disease. Alterations in the basic equilibrium constituted by tissue plasminogen activator (t-PA) and plasminogen activators inhibitor (PAI) have been implicated in arterial thrombosis (7, 8, 9). Elevated levels of PAI- have been described in patients with chronic coronary artery disease without a history of previous myocardial infarction (lo), in young survivors of myocardial infarction (11) and associated to increased risk of reinfarction (12). Coronary lesion significantly correlated with total cholesterol and inversely with HDL-cholesterol levels (13) Besides, high concentration of lipoprotein(a), Lp(a), has been demostrated to be associated with an increased risk of cardiovascular disease (14, 15, 16, 17, 18) and, recently , it has been documented in hypercholesterolemic patients that Lp(a) level is the best discriminant among lipidic parameters between those with and those without ischaemic heart disease (19, 20). Apoprotein (a) has strong structural similarities with plasminogen (21, 22) and, actually, it has been demonstrated that competes with plasminogen "in vitro" for the binding to Lp(a) fibrin (23, 24) fibrinogen (24. 25) and cell membrane binding sites (26, 27). Thereby, the presence of Lp(a) could result in a less efficient activation of plasminogen to plasmin, by interfering with the fibrinolytic system. Therefore, the ratio between Lp(a) and plasminogen or other fibrinolysis related substances may be of interest for evaluation of the cardiovascular risk, The aim of this work was to evaluate the fibrinolytic system and relationship lipoprotein(a) in its with patients with chronic angina in a stable condition and after myocardial infarction as two different stages in coronary artery disease. PATIENTS AND METHODS Thirty four patients with chronic angina pectoris with more than a year of evolution, age median 60 years (range from 48 to males and 2 females, and 12 patients with acute 7o), 32 myocardial infarction (AMI) 15-20 days after the acute episode, age median 63 years (range from 58 to 63), 10 males and 2 females were studied. A coronariography was performed on all of them. A group of 31 healthy blood donors similar in age and sex were chosen as controls. Blood samples were obtained by venipuncture, in a fasting state and after a supine rest of at least 15 min. between 8 and

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10 a.m. Poststasis blood samples were obtained after 10 min. of pressure between minimum and maximun. Treatment was discontinued 4 days in advance. Platelet poor plasma (PPP) was obtained by centrifugation at was 3000 g for 15 minutes. The PPP aliquoted and stored at -70C. Fibrinogen (Fg) was measured by thrombin time assay (28); alpha*-antiplasmin (AP), and plasminogen (Pg) were determined by a Laurel1 rocket immunoelectrophoretic technique (29), using rabbit antihuman antibodies (Behring Institute). Tissue plasminogen activator (t-PA) was evaluated by an enzyme linked immunospecific assay (ELISA) according to Rijken et al. (30) with the exception that all 96 well polyvinyl plates were precoated with goat IgG t-PA antibodies and stored during the study at -40C. Each subsequent step was confined to 1 hour incubation at 37C allowing three hours approximately to perform the ELISA procedure. Goat IgG (anti-rabbit IgCi)conjugated with alkaline phosphatase was obtained from Calbiochem (La Jolla, USA), diethanolamine, bovine albumin from Sigma (St. Louis, USA), and Tween 80 from Merck (Schuchardt, Darmstadt, FRG). The samples were compared with the International Standard for t-PA (National Institute for Biological Standards and Control, Hert., England) which defines the international unit of t-PA as being equivalent to approximately 2 ng of the ampouled preparation of t-PA (ampoule code 83/517). TPA/PAI complexes were assayed by an ELISA assay using monoclonal antibodies. A t-PA-PA1 complex was used as standard. EDTA was previously added to samples up to a final concentration of 5mM. (Technoclone T-PA-PAI-COMPLEX ELISA, Vienna, Austria). Plasminogen activator inhibitor (PAI) was measured with a monoclonal antibody based ELISA (31) (TintElize PAI-1, catalog number 210220, Biopool AB Umea, Sweden). A fibrin plate (FP) was done according to Haverkate and Traas (32) using agarose (2.5%) and human fibrinogen (10.0 mglml). using Fibrin degradation products assay was performed monoclonal antibodies to D-dimer fibrin fragments in an ELISA assay (Boehringer Mannheim, Germany) (33). Cholesterol and triglycerides were determined by enzymatic method kits (Boehringer Mannheim GmbH Diagnostica, Mannheim, and plasma Germany), HDL-cholesterol was separated by precipitation with acid-MgCl, (Boehringer phosphotungstic Mannheim, Germany). LDL-cholesterol was estimated according to the Friedewald formula. Plasma levels of lipoprotein(a) were analysed by an ELISA assay using polyclonal antibodies (Tintelize Lp(a), Biopool AB Umea, Sweden). Data are given as median with interquartile range. The Mann-Whitney test was used for comparison between groups. A p value of 0.05 or less was considered as statistically significant. Correlation coefficients were calculated using the Pearson and Spearman Rank tests.

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RESULTS All patients had at least one significant coronary artery stenosis i>75%) in the angiography: i2 patients had one vessei disease, 18 two and 16 three vessel disease. As risk factors, sedentarism was present in 33 (72%) patients, tobacco in 38 hypertension in 8 (lg%), diabetes in 8 (19%) and (83%). overweight (body-mass index>25) in 26 (57%). In table I the lipids and plasma Lp(a) concentration of the coronary artery disease patients studied are shown. Cholesterol, LDL-cholesterol. triglycerides and levels were Lp(a) significantly increased and HDL-cholesterol was decreased in angina related to controls. Lp(a) was the only parameter that was significantly different in AM1 patients compared to controls. No differences were found in Lp(a) levels between AM1 and angina patients. In fig 1 distribution of plasma Lp(a) concentrations in coronary artery disease patients (AM1 and angina patients considered as a whole group) related to controls is shown. It must be pointed out that while only 11.5% of controls have Lp(a) concentration higher than 30 mg/dl, 53% of coronary arterial disease patients surpass that level. Usinn Svearman Rank test no correlation was found between Lp (a) p&ma- .evels and the degree of vessel disease. Distributionof plasma Lp(a) I~‘~!‘~~!~”

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Lipoprotein(a) (mg/dl) Figure 1. Frequency distribution of plasma lipoprotein(a) in controls (n=31) and CAD patients (n=46) (CAD group corresponds to both AM1 and angina patients).

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TABLE I

Serum Lipids and Lp(a) of 46 Patients with Coronary Artery Disease CONTROL(n=31) ANGINA(n=34) AMI(n=12) 206 (204-221) 249(199-287)” Cholesterol(mg/dl) 213 (193-247) 152(123-190)" Triglycerides(mg/dl) 110 (95-147) 107 ( 97-148) HDL-Chol.(mg/dl) 42 ( 36- 50) 37( 31-42)" 42 (36-52) 147 (125-166) LDL-Chol.(mg/dl) 139 (135-148) 174(129-211)" 35.9 (6.5-62.4)" 24.7(8.1-56.6)"" 10.5(5.2-14.9) Lp(a)(mg/dl) *p

Fibrinolytic parameters and lipoprotein (a) levels in plasma of patients with coronary artery disease.

Fibrinolysis and lipid disturbances have been considered as independent risk factors for coronary artery disease. Besides this, lipoprotein(a), which ...
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