Fibrin Clot Properties and Fibrinolysis in Patients With Endocrine Hypertension Due to Aldosterone or Catecholamines Excess

Ewa Warchoł-Celińska; Aleksander Prejbisz; Piotr Dobrowolski; Ewa Wypasek; Jacek Kądziela; Sylwia Kołodziejczyk-Kruk; Marek Kabat; Anetta Undas; Andrzej Januszewicz


Clin Endocrinol. 2022;96(2):114-122. 

In This Article

Materials and Methods

Our study included 35 patients with PA (Group 1), 16 patients with PPGL (Group 2), two control groups of patients with EHT—32 patients (Control group 1) and 22 patients (Control group 2), respectively and two control groups of healthy controls—35 subjects (Control group 1bis) and 23 subjects (Control group 2bis). Both control groups of EHT patients were matched with PA and PPGL groups (Control group 1 and Control group 2, respectively) for age, gender, severity of hypertension, number and distribution of antihypertensive agents, as well as cardiovascular risk factors, including hypercholesterolaemia, glucose profile, metabolic syndrome and smoking. Both control groups of healthy controls were matched with the group of PA and PPGL groups (Control group 1bis and Control group 2bis, respectively) for age, gender and body mass index (BMI). All patients with PA, PPGL and EHT were hospitalized in the Department of Hypertension, National Institute of Cardiology, Warsaw, Poland in 2014–2017 (Figure 1). The reference group of healthy controls were established from well-matched medication-free and apparently healthy subjects assessed in the Innovative Laboratory Diagnostic Centre, John Paul II Hospital.

Figure 1.

Study flow chart. APA, aldosterone-producing adenoma; BAH, bilateral adrenal hyperplasia; EHT, essential hypertension; PA, primary aldosteronism; PPGL, pheochromocytoma and paraganglioma

Inclusion criteria were as follows: (1) age 18–65 years, (2) Study group 1—biochemically proven PA according to Management of Primary Aldosteronism: Case Detection, Diagnosis and Treatment: An Endocrine Society Clinical Practice Guidelines, (3) Study group 2—biochemically and histopathologically proven catecholamine producing tumour(s) according to Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline, (4) Control groups 1 and 2—essential hypertension (excluding PA, PPGL renal artery stenosis, Cushing's syndrome, subclinical Cushing's syndrome);[13,14] (5) Control group 1bis and 2bis—healthy subjects, with no pharmacological treatment and no signs of acute infection, or recent trauma or surgery.

Subjects with established cardiovascular or renal disease, known cancer and pregnancy were excluded. Subjects receiving anticoagulant therapy or with a history of deep vein thrombosis or pulmonary embolism were also excluded from the study. The detailed exclusion criteria for all groups are listed in the Supporting Information. In all patients with PPGLs and PA, the diagnostic and treatment decisions were based on the current guidelines.[13,14] The study was approved by the Ethics Committee of the National Institute of Cardiology, Warsaw, and informed written consent was obtained from each patient.

All included patients underwent meticulous evaluation according to the study protocol (physical examination, office and 24-h BP measurement, standard laboratory evaluations, inflammatory markers, hormonal evaluation, evaluation of fibrin clot properties). Patients with PA and PPGL were re-evaluated 3 months after surgical treatment—patients with APA, PPGL or 3 months after the introduction of mineralocorticoid antagonist (MRA)—patients with BAH. At 3 months, hormonal evaluation was performed in patients who had undergone surgery (patients with APA, PPGL; Figure S1). The biochemical and clinical outcome after adrenalectomy in APA patients was evaluated according to the PASO study protocol and defined as complete or partial clinical and biochemical success.[15]

Clinical Evaluation and BP Measurements

All patients underwent a physical examination. Medical history, concomitant medication and general characteristics (date of birth, age, gender, BMI, waist circumference and ethnic background) and current pharmacological treatment including antihypertensive agents were recorded. Hypertension was defined as BP ≥ 140/90 mmHg on office measurements or current antihypertensive medication. The methodology of office and 24-h BP measurements is presented in the Supporting Information.

Laboratory Evaluation

All blood samples were taken after overnight fasting. The detailed methodology of routine blood tests is described in the Supporting Information. Citrated plasma samples were stored at −80°C until analysis. All measurements were performed by technicians blinded to the sample status. Immunoenzymatic and chromogenic assays were used to determine haemostatic markers: tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), plasma-activated thrombin-activatable fibrinolysis inhibitor (TAFI), prothrombin fragments 1 + 2 (F1.2), plasmin-alpha-2-antiplasmin complex (PAP). The methodology is described in the Supporting Information. The measurement of clot properties presented in the Supporting Information involved determination of plasma fibrin clot permeability (Darcy's constant, Ks), as a measure of an average pore size in fibrin networks, and clot lysis time (CLT) as a measure of t-PA-induced degradation of fibrin clots generated in the presence of tissue factor and phospholipids. We measured thrombin generation with the use of calibrated automated thrombography and its kinetics profile was presented using the following variables: lag time—time from the beginning of reaction to the beginning of thrombin generation, ETP—endogenous thrombin potential, that is, area under the thrombin-generation curve (total amount of thrombin generated during the test); thrombin peak—peak thrombin generation and ttPeak—time to peak thrombin generation.

Evaluation of Secondary Forms of Hypertension

The detailed methodology of the diagnostic scheme for PA including saline infusion test, the determination of PA subtype with the use of adrenal veins sampling (AVS), is presented in the Supporting Information. Biochemical testing for PPGL included mass spectrometric-based measurements of catecholamine metabolites (normetanephrine, metanephrine, methoxytyramine) in their free unconjugated form in plasma (see Supporting Information). All other data including blood collections, reference intervals and designations of tumours have been described elsewhere.[16,17]

Statistical Analysis

Data analysis was carried out using the statistical software PASW Statistics 18 (SPSS Inc). The methodology of statistical analysis is presented in the Supporting Information.