Relationships between coagulation factors and thrombin generation in a general population with arterial and venous disease background

Research design

The Gutenberg Health Study (GHS) is a prospective, observational, single center cohort study, designed for population-based health research, in the Rhine-Main region in Germany. With a total of 15,010 individuals between 35 and 74 years enrolled at the baseline examination, the GHS aims to assess the consequences of diseases and environmental factors in addition to the inherited predisposition on the development and progression of asymptomatic and symptomatic disease. During the baseline visit, every participant underwent a comprehensive, standardized 5-hour clinical examination program, as reported elsewhere [16, 17]. The baseline visit at the GHS study centre comprised a standardized 5-h investigation according to standard operating procedures. Participants underwent a detailed computer-assisted interview covering assessment of cardiovascular risk factors, lifestyle, socioeconomic status, and other areas. The prevalence of cardiovascular disease was determined by history taking. In addition to the extensive clinical assessment, a large biobank has been established for future biochemical and genetic analyses. As part of the follow-up, a standardized computer-assisted telephone interview and an inventory of primary and secondary endpoints were done 2.5 years after baseline visit. In addition, participants undergo a quinquennial, extensive clinical examination in the same research facility as the baseline visit. Primary endpoints of the study were myocardial infarction and cardiovascular death. Secondary endpoints were cerebrovascular accident, diabetes mellitus, heart failure, atrial fibrillation or death caused by the previously named diseases. Details of the study protocol and the further purposes of the study are discussed elsewhere [18].

Study sample

From the initial GHS cohort including 5000 subjects TG data was available for 4843 subjects. From these, 1402 individuals were included in the reference group, 502 in the arterial disease group, and 195 in the venous disease group. Selection for each group was as follows:”

The overall study sample consisted of the first 5000 subjects enrolled into the GHS between April 2007 and October 2008. After excluding subjects without biomaterial available or without complete TG assessment (one or several TG parameters were missing), 4843 individuals were successfully included in the present analysis.

The reference group was defined as apparently cardiovascular healthy subjects without history of cardiovascular diseases (myocardial infarction [MI], congestive heart failure [CHF], coronary artery disease [CAD], peripheral artery disease [PAD], venous thromboembolism [VTE], atrial fibrillation [AF]), presence of cardiovascular risk factors (CVRF; obesity, dyslipidemia, arterial hypertension, diabetes mellitus) and included 1402 individuals. Individuals with a self-reported history of inherited coagulation abnormalities were excluded from the reference sample. The arterial disease group was defined as individuals with a history of MI, CAD, stroke or PAD and included 502 individuals. The venous disease group was defined as individuals with a history of deep venous thrombosis (DVT) or pulmonary embolism (PE) and included 195 individuals. Individuals that did not meet the above-mentioned criteria of the various groups were excluded from the current analysis. For a detailed definition of traditional CVRF and categorization of medications, please see Supplemental Material, Part A.

Blood sampling and laboratory assessment

Venous blood sampling was performed according to standard operating procedures and the blood was collected in trisodium citrate (0.109 M, 1:9 vol:vol) monovette plastic tubes, while the subject was in fasting state (i.e. overnight fast, if subject was examined before 12 p.m.. and 5 hour fast, if subject was examined after 12 p.m.). Platelet poor plasma (PPP) was prepared by one-step centrifugation at 2000 x g at room temperature for 10 minutes. After preparation the PPP was aliquoted and immediately stored at − 80 °C in the Biobank of the GHS study center.

The TG was assessed in the Laboratory for Clinical Thrombosis and Hemostasis, Maastricht University, the Netherlands, by the Calibrated Automated Thrombogram (CAT) assay (Thrombinoscope BV, Maastricht, The Netherlands), according to the recommendations [19, 20]. The TG was triggered by PPP Reagent Low (Stago) in freshly thawed PPP. The CAT method employs a low affinity fluorogenic substrate for thrombin (Z-Gly-Gly-Arg-AMC) to continuously monitor thrombin activity 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–alfa 2-macroglobulin complex (20 μL of Thrombin Calibrator; Thrombinoscope BV, Maastricht, The Netherlands) and 20 μL of the fluorogenic substrate and calcium chloride mixture. TG parameters were derived from the TG curve and include lag time (time to minimum thrombin formed [min]), peak height (the maximum amount of thrombin formed [nM]) and endogenous thrombin potential (ETP or area under the curve [nM.min]).

Coagulation factors were measured by means of BCS XP Systems in the Biomolecular laboratory at the Department of Epidemiology, University Medical Center Mainz, Germany. The coagulation factors II, V, VII, VIII, IX, X, XI, XII were determined using the clotting-based coagulation methodology, protein C and antithrombin by the chromogenic assay and von Willebrand factor (vWF) and protein S by using immunological-based assay. Reference values by the WHO standard provided by Siemens were used. Total TFPI activity was assessed in PPP by the Actichrome TFPI activity assay (American Diagnostica, Stamford, CT, USA) in the Laboratory for Clinical Thrombosis and Hemostasis, Maastricht University, the Netherlands.

Data management and statistical analysis

A central data management unit conducted quality control on all data in this study. Statistical analysis was performed with software program R, version 3.3.1 (http://www.R-project.org). Data on coagulation factors and inhibitors are presented as mean (standard deviation) in case of normal distribution.

Multiple linear regressions were used to assess the associations between biochemical variables and TG parameters in the reference group as well as in the arterial and venous disease group. The analyses were adjusted for age, sex and additionally for hormones (oral contraceptives and hormone replacement therapy = G03) and anti-coagulant agents (B01AA, B01AB, B01AE, B01AF, B01AX) as these may affect the thrombin potential. Due to a skewed distribution, lag time, as a dependent variable, was log-transformed prior to the analysis. Estimated beta regression coefficients, presented with corresponding 95% confidence interval (CI), were calculated as per standard deviation to compare the effects of different coagulation-related factors on TG parameters. Due to its explorative nature, a p-value threshold was not defined. However, to account for multiple statistical tests and minimize the risk of type 1 error, a Bonferroni corrected p-value (0.00036) was set for the results on the multiple linear regression analyses.

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