J Am Soc Nephrol 12: 1255–1263, 2001 Circulating Levels of Plasminogen Activator Inhibitor Type-1, Tissue Plasminogen Activator, and Thrombomodulin in Hemodialysis Patients: Biochemical Correlations and Role as Independent Predictors of Coronary Artery Stenosis ALFONS SEGARRA,* PILAR CHACON,´ † CRISTINA MARTINEZ-EYARRE,‡ XAVIER ARGELAGUER,* JOSEFA VILA,* PILAR RUIZ,* JOAN FORT,* JORGE BARTOLOME,*´ JOAQUIN CAMPS,* ERNESTO MOLINER,§ ANTONI PELEGR´I,࿣ FERNANDO MARCO,¶ ANTONIO OLMOS,* and LLUIS PIERA* *Servicios de Nefrologı´a and †Bioquı´mica, Hospital Valle Hebro´n, Barcelona; ‡CDR Monolab, §Unidad de Hemodia´lisis, Hospital Sant Gervasi; ࿣Centro de Nefrologı´a, Virgen de Montserrat; and ¶Centro de Dia´lisis Nephros, Barcelona, Spain. Abstract. This study investigated the relationship between the extent with major classic vascular risk factors and to a lesser circulating levels of the endothelial cell glycoproteins plasmin- extent with CRP and serum triglycerides. Forty-six patients ogen activator inhibitor type 1 (PAI-1), tissue plasminogen (23%) had evidence of CAD. Variables associated with CAD activator (TPA), and thrombomodulin (TM) and the major in the univariate analysis included age, time on dialysis, male vascular risk factors described in dialysis patients. In addition, gender, number of packs of cigarettes per year, high BP, the role of these endothelial cell products as independent fibrinogen, apolipoprotein B, albumin, PAI-1 activity, CRP, predictors of coronary artery disease (CAD) was analyzed. thrombin-antithrombin complexes, and fibrinopeptide A. Lo- Levels of TM, TPA antigen (Ag), TPA activity, PAI-1 Ag, gistic regression analysis found age, high-density lipoprotein PAI-1 activity, TPA/PAI complexes, thrombin-antithrombin cholesterol, gender, high BP, CRP, time on dialysis, and PAI-1 complexes, fibrinopeptide A, C-reactive protein (CRP), inter- activity to be independent predictors of CAD. This model leukin-1␤ and tumor necrosis factor-␣, lipids, apoproteins A1 classified correctly 85% of patients as having CAD and and B, and albumin were measured in a group of 200 nondi- showed adequate goodness of fit for all risk categories. Our abetic dialysis patients and 100 healthy matched volunteers. data support a pathogenic link among activated inflammatory When compared with healthy controls, dialysis patients response, endothelial injury, and CAD in hemodialysis patients showed increased levels of CRP, TM, TPA, and PAI-1 and and suggest that assessment of circulating PAI-1 levels could evidence of increased thrombin-dependent fibrin formation. be an additional tool to identify dialysis patients who are at risk Increased levels of active PAI-1 were associated to a great for developing atheromatous cardiovascular disease. Central to the response-to-injury hypothesis is the proposal that vidual patients, some of them are elevated significantly in the different vascular risk factors somehow lead to endothelial disorders with acute endothelial damage and may provide cell injury, which can elicit a series of cellular interactions that reliable correlations in large population studies (3–8). In recent culminate in the lesions of atherosclerosis (1). Several endo- years, it has been shown clearly that circulating levels of PAI-1 thelial products have been proposed as possible in vivo markers and other endothelial cell glycoproteins are increased in hemo- of the endothelial cell injury, such as von Willebrand factor, dialysis patients (9–13). Although this increase has been con- thrombomodulin (TM), tissue plasminogen activator (TPA), sidered as a subclinical sign of endothelial cell injury (9,12), plasminogen activator inhibitor (PAI), and soluble p-selectin this association is merely a hypothesis that remains to be (2–5). Although all of these markers lack sensitivity and/or proved. Before the circulating levels of PAI-1 and other endo- specificity for assessment of endothelial dysfunction in indi- thelial glycoproteins are considered as indicators of a chronic endothelium activated state, other potential mechanisms that contribute to the increased levels of these endothelial products Received July 19, 2000. Accepted November 1, 2000. should be taken into account. First, it is widely known that Correspondence to Dr. Alfons Segarra Medrano, Unidad de Investigacio´n, PAI-1 may behave as an acute-phase reactant (14). Second, Servicio de Nefrologı´a, Hospital Valle Hebro´n, Passeig Vall d’Hebro´n, 119- certain studies suggest that PAI-1 activity in chronic renal 129, E-08035 Barcelona, Spain. Phone: 34-93-274 61 52; Fax: 34-93-274 62 04; E-mail: [email protected] failure and dialysis patients is associated strongly with the 1046-6673/1206-1255 common metabolic abnormalities of obesity and hyperlipid- Journal of the American Society of Nephrology emia (15). Moreover, if circulating endothelial glycoproteins Copyright © 2001 by the American Society of Nephrology such as PAI-1, TPA, and TM are subclinical markers of endo- 1256 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 1255–1263, 2001 thelial cell injury, then the levels of these molecules should be PAI-1 activities were determined by chromogenic assays (Biopool, statistically associated with major classical vascular risk fac- Umea, Sweden). Fibrinopeptide A (FPA) was determined by ELISA tors and/or with the presence of atheromatous cardiovascular (Boehringer Mannheim GmbH; Diagnostica Stago). C-reactive pro- disease. tein (CRP) was determined by the Behring Nephelometry immuno- This study was designed (1) to investigate the relationship assay (NA latex CRO, Behring Institute, Galway, Ireland). Serum interleukin-1␤ (IL-1␤) and tumor necrosis factor-␣ (TNF␣) were between the circulating levels of the endothelial cell glycop- determined by ELISA (Medgenix Diagnostics, Brussels, Belgium). roteins PAI 1, TPA, and TM and the major vascular risk factors Albumin concentration was determined by the green bromcresol described in dialysis patients and (2) to determine the role of method. these endothelial cell products as independent predictors of All assays were performed in duplicate and calibrated with purified CAD in a large group of nondiabetic dialysis patients. standards and reference plasmas from the manufacturers. Intra- and interassay coefficients of variation for all tests were determined with Materials and Methods the use of 20 different plasma samples. Patients To avoid the influence of acute intercurrent diseases on the bio- chemical parameters, we carried out analyses at least 6 mo after the We studied 200 nondiabetic patients who were receiving dialysis clinical event in all patients who required hospitalization for acute treatment in three outpatient dialysis centers affiliated with our ne- illness or infections (n ϭ 6) or who had surgical procedures (n ϭ 4), phrology department. The study group consisted of 120 men and 80 episodes of kidney allograft rejection (n ϭ 2), or acute vascular women, 31 to 80 yr old, dialyzed three times a week for 4 to 200 mo. thrombosis (n ϭ 1) before being entered in the study. The hemodialysis prescription was 9 to 13.5 h/wk, with the use of a A prerequisite for including the levels of endothelial cell glycop- 1.2- to 1.6-m cuprophane hollow-fiber filter and bicarbonate dialysate roteins, CRP, and inflammatory cytokines in statistical analyses was containing 2 g/L glucose. The dialyzers were not reused. All patients to demonstrate that the predialysis levels of all of these variables received a multivitamin supplement after the dialysis sessions, includ- showed little variation over time when measured in the same group of ing vitamins C and B and folic acid. A total of 140 patients (70%) patients. For this purpose, we analyzed the variation coefficients for received erythropoietin therapy, 70 patients (35%) received antiplate- the different variables in 30 random patients before the start of four let drugs (aspirin or ticlopidine), 65 patients (32.5%) were treated with consecutive dialysis sessions. They were as follows: TPA Ag, 12%; angiotensin-converting enzyme inhibitors (ACEI), and 34 patients PAI-1 Ag, 16%; PAI-1 activity, 14%; FPA, 8.4%; TAT complexes, (17%) received therapy with simvastatin or pravastatin. 12.3%; CRP, 11%; IL1-␤, 26%; TNF-␣, 20%; and soluble TM, 9.8%. The cause of chronic renal failure was glomerular disease in 70 patients (35%), interstitial nephritis in 30 patients (15%), polycystic kidney disease in 35 patients (17.5%), vascular disease in 20 patients Clinical Data Collection (10%), and unknown in 45 patients (22.5%). Information on risk factors was obtained by medical record review, The control group comprised 100 healthy, age- and gender- personal interview, and physical examination. Coronary angiography matched volunteers who were recruited from among the population of was performed on all patients who had no previous episodes of healthy people living in the geographic area of our center. well-documented acute myocardial infarction and who experienced The study protocol was accepted by the ethics committee of our clinical symptoms that suggested ischemic heart disease associated hospital, and all patients gave their written informed consent before with ST changes in the ECG. participation. Definitions Laboratory Procedures CAD was diagnosed in the presence of one of the following: (1) For lipid and apoprotein assays, blood was collected from the definitive episode of myocardial infarction with appropriate rise in antecubital vein in glass tubes with no additives after an overnight