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P.C.S. van Paridon et al.
This large-scale study demonstrates that traditional cardiovascular risk factors, particularly obesity, are relevant determinants of thrombin generation. Lag time and endogenous thrombin potential were found to be potentially relevant predictors of increased total mortality, observations which deserve further investigation.
Introduction
Thrombin generation (TG) is one of the key enzymatic processes that direct the activity of the hemostatic system and coagulation cascade up to and including the formation of a fibrin clot.1 Physiologically, thrombin formation is essential to maintain hemostasis and bleeding tendencies are associated with reduced thrombin (and hence fibrin) formation.2 An enhanced plasma potential to generate thrombin has been linked to an increased risk of venous thromboembolism, while the associations with arterial vascular disease are still inconsistent.3-8 The TG assay is an important method addressing the overall potential of a plasma sample to form thrombin. More than 95% of TG occurs after initial formation of fibrin, so routine diagnos- tic coagulation tests, such as prothrombin time and acti- vated partial thromboplastin time fail to reproduce this overall potential. Hence, there is a strong research interest in TG as a promising diagnostic tool for hypo- and partic- ularly hyper-coagulability phenotypes.9
In a study of healthy individuals, fibrinogen, factor XII, free tissue factor pathway inhibitor (TFPI) and antithrom- bin have been identified as major determinants of TG parameters.10 Relation to demographic characteristics, such as age and sex, has been previously addressed but the studies have been small and results are not entirely consis- tent.11,12 As TG analysis is a promising tool to estimate a subject’s risk for thrombosis or more broadly cardiovascu- lar diseases (CVD), it is of eminent importance to fully understand the nature and direction of effects of cardio- vascular risk factors (CVRF).
Hence, we undertook the present investigation in the first 5,000 participants of the population-based Gutenberg Health Study. The primary aim of this study was to inves- tigate CVRF and CVD as major clinical determinants of increased TG in a large population-based sample. Additional aims were to obtain age- and sex-related refer- ence values for TG parameters in a representative subsam- ple of adults who were healthy from a cardiovascular point of view. Finally, having prospective data on total mortality allowed us to investigate the relation between TG parameters and all-cause mortality.
Methods
Research design
The Gutenberg Health Study, a population-based, prospec- tive, observational, single-center cohort study in the Rhine-Main region in Western Mid-Germany, was designed to improve the individual risk prediction of CVD. At baseline examination, the study included a total of 15,010 individuals. A detailed descrip- tion of the research design is provided in Online Supplementary Material, Part A. Further details of the study protocol and pur- pose are discussed elsewhere.13
The study was designed in accordance with the tenets of the revised Helsinki protocol, and the protocol and sampling design
were approved by the local ethics committee. The sampling design was additionally approved by local and state data safety commissioners.
Study sample and reference sample
The study sample consisted of the first 5,000 subjects enrolled into the Gutenberg Health Study between April 2007 and October 2008. After excluding subjects without biomaterial available or without complete TG assessment (one or several TG parameters missing), 4,843 individuals were successfully included in the overall study sample for the present analysis.
The reference group was defined as subjects apparently healthy from a cardiovascular point of view, without a history of CVD (myocardial infarction, congestive heart failure, coro- nary artery disease, venous thromboembolism, atrial fibrillation or peripheral artery disease), presence of CVRF (obesity, dyslipi- demia, arterial hypertension, diabetes mellitus) or use of antithrombotic agents, oral contraceptives or hormonal replace- ment therapy. In addition, individuals with a self-reported histo- ry of inherited coagulation abnormalities were excluded from the reference sample. A detailed definition of traditional CVRF and categorization of medications are provided in Online Supplementary Material, Part A.
Clinical assessment and laboratory measurements
Clinical examination and determination of CVRF were per- formed as published elsewhere.14,15 Standard laboratory meas- urements were carried out at the Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Mainz, Germany. Details on venous blood sampling and plasma prepa- ration are provided in Online Supplementary Material, Part A. TG was assessed according to the recommendations16 for the Calibrated Automated Thrombogram (CAT) assay (Thrombinoscope BV, Maastricht, the Netherlands) in platelet- poor plasma. The TG was triggered by 1 pM tissue factor (TF) with 4 μM phospholipids at 20:20:60 mol% phosphatidylser- ine/phosphatidylethanolamine/phosphatidylcholine, or 5 pM TF with 4 μM phospholipids. Trigger reagents were selected for commercial availability, e.g. PPP Reagent and PPP Low Reagent. The CAT method employs a low affinity fluorogenic substrate for thrombin (Z-Gly-Gly-Arg-AMC) in order to monitor throm- bin activity continuously in clotting plasma. TG measurements were calibrated against the fluorescence curve obtained in a sample from the same plasma (80 μL), supplemented with a fixed amount of thrombin-a 2-macroglobulin complex (20 μL of Thrombin Calibrator; Thrombinoscope BV, Maastricht, the Netherlands) and 20 μL of the fluorogenic substrate and calcium chloride mixture.16 TG parameters were derived from the TG curve and include lag time (time to minimum thrombin formed, in min), peak height (the maximum amount of thrombin formed, in nM) and endogenous thrombin potential (ETP or area under the curve, in nM.min). All samples were tested as one batch using one batch of reagents within a period of 6 months. Two technicians performed the analyses on three vali- dated systems and normal pooled plasma was included in each assay run for in-house quality control according to our ISO9001 certification (Coagulation Profile BV, Maastricht, the Netherlands).
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haematologica | 2020; 105(9)