Objective
Pregnancy is a hypercoagulable state. We evaluated global markers of coagulation activation, ProCGlobal (Siemens Healthcare Diagnostics, Eschborn, Germany) and endogenous thrombin potential (ETP), in pregnant women with and without low-molecular-weight (LMW) heparin prophylaxis.
Study Design
We prospectively followed 113 healthy women and 61 women receiving LMW heparin prophylaxis throughout pregnancy. ProCGlobal and ETP were measured in venous blood during the first, second, and third trimester.
Results
ProCGlobal decreased significantly throughout pregnancy in all women and was lower in anticoagulated women ( P < .001 for all comparisons). ETP levels remained unchanged until the third trimester and then significantly decreased in all women. ETP was higher in anticoagulated women than in healthy women at all time points.
Conclusion
ProCGlobal levels decrease throughout pregnancy. In pregnant women at high thrombotic risk, coagulation activation reflected by low ProCGlobal and high ETP levels is substantial despite LMW heparin prophylaxis.
Pregnancy is associated with alterations of the hemostatic system. The plasma concentrations of procoagulant factors including factors VII, VIII, IX, X, XII, fibrinogen, von Willebrand factor, as well as microparticles increase, while the activity of anticoagulant and fibrinolytic factors including protein S and plasminogen activator decreases during pregnancy. Overall, these changes induce a hypercoagulable state, which is reflected by a shortened activated partial thromboplastin time (aPTT) and elevated markers of coagulation activation. In healthy women the levels of D-dimer, a fibrin degradation product, and prothrombin fragment F1.2, which is cleaved from prothrombin after its activation to thrombin, substantially increase throughout pregnancy and return to baseline levels several weeks after delivery. In women at risk of thrombosis, pregnancy is accompanied by a significant activation of the coagulation despite low-molecular-weight (LMW) heparin thromboprophylaxis.
Recently, novel assays that can be used for a more global assessment of coagulation activation have been developed. The ProCGlobal (Siemens Healthcare Diagnostics, Eschborn, Germany) assay tests the global function of the protein C pathway after coagulation activation by a viper venom from Agkistrodon contortrix. The assay is suitable to detect hereditary or acquired defects in the protein C system, including protein C and protein S deficiency, resistance to activated protein C, and the factor V Leiden mutation. In a prospective cohort study, ProCGlobal was useful to stratify patients with a first unprovoked venous thromboembolism (VTE) according to their risk of recurrence. ProCGlobal assay has, however, not been evaluated during pregnancy.
The capacity of in vitro thrombin generation is related to the risk of venous thrombosis. Thrombin generation can continuously be registered by measuring cleavage of a slow-reacting thrombin substrate. Thrombin generation over time follows a characteristic curve and the calculated area under this curve is called the endogenous thrombin potential (ETP). Thus, ETP provides a measure of the thrombin-mediated capacity of the plasma. Data on ETP during pregnancy are limited and results are conflicting.
It was the aim of the present study to evaluate the ProCGlobal assay and the ETP in a prospective cohort of healthy pregnant women and in women who received LMW heparin thromboprophylaxis during pregnancy.
Materials and Methods
Study design and study population
The present study is part of a prospective cohort study in which women (age ≥ 19 years) who were otherwise healthy were followed up throughout pregnancy. Women were recruited during the first trimester and were excluded in case of 1 of the following conditions: a pregnancy-associated disease (eg, hypertension, preeclampsia/eclampsia), treatment with antithrombotic drugs, a systemic disease (eg, diabetes, malignancy, antiphospholipid syndrome), or a history of venous or arterial thrombosis. The study was approved by the Ethics Committee of the Medical University of Vienna, Austria, and all women gave written consent to participate.
We also prospectively studied another cohort of pregnant women who required anticoagulant treatment with LMW heparin for ≥1 of the following reasons: a history of VTE, hereditary thrombophilia, or previous pregnancy-related complications (preeclampsia/eclampsia, abortion, stillbirth, or placental abruption). Details of the study have been previously reported. Women were treated with either enoxaparin or dalteparin at a dosage of 4000-7500 antifactor Xa IU subcutaneously. The dosing was left to the discretion of the physician. Treatment was started as soon as the pregnancy was confirmed and was continued throughout pregnancy.
Follow-up and blood sampling
Women with and without anticoagulation were seen during their first (12th ± 3 gestational week), second (22nd ± 3 gestational week), and third (34th ± 3 gestational week) trimester at the Department of Internal Medicine I, Medical University of Vienna, or at the Department of Gynecology and Obstetrics, Donauspital, Vienna, Austria. At each visit, a medical history was obtained and venous blood samples were collected into 0.1-mmol/L trisodium citrate vacutainer tubes. Platelet poor plasma was obtained by after centrifugation (1600 g , 4°C, 15 minutes) and stored at –80°C until batch analysis.
Laboratory analysis
ProCGlobal was performed by use of a commercially available assay (ProC Global, Siemens Healthcare Diagnostics, Eschborn, Germany) on blood coagulation system (BCS XP, Siemens Healthcare Diagnostics) analyzer. This assay is based on the aPTT, which is determined before (protein C activation time [PCAT]-0) and after activation of plasma protein C by Protac, a snake venom (PCAT). For PCAT-0, 50 μL of aPTT reagent and 50 μL of buffer were added to 50 μL of plasma. For PCAT, 50 μL of aPTT reagent and 50 μL of Protac were added to 50 μL of plasma. After incubation for 3 minutes at 37°C samples were measured. Values are given as a normalized ratio (NR) by dividing the PCAT ratio (PCAT/PCAT-0) of the sample plasma by a PCAT ratio of lyophilized standard human plasma and multiplying by a lot-specific sensitivity value defined for each batch by the manufacturer.
ETP was determined in platelet poor plasma by a commercially available (for research use only) assay (Siemens Healthcare Diagnostics). Coagulation activation was initiated by incubation of plasma with phospholipids, human recombinant tissue factor (Innovin, Siemens Healthcare Diagnostics), and calcium ions in the absence of thrombomodulin. The concentrations of phospholipids and tissue factor are confidential to the manufacturer.
Thrombin generation was recorded by monitoring conversion of a specific slow-reacting chromogenic substrate at a wavelength of 405 nm over time. A mathematical algorithm was applied to correct the substrate conversion curve for the activity of α2-macroglobulin bound thrombin, which has no known biological activity, but is still capable of cleaving small chromogenic substrates. The ETP value was calculated as the area under the thrombin generation curve. Evaluation of reaction curves as well as computer-assisted calculation of thrombin generation over time was done by the curve evaluation software Curves, Version 1.0 with specification 3.2. (Siemens Healthcare Diagnostics, for research use only). ETP values are given as percent (%) of normal. Standardization was done by measuring the ETP standard (Siemens Healthcare Diagnostics) daily in parallel to the patient samples. Value assignment (% of normal) of the ETP standard was performed against a normal plasma pool of 80 healthy donors that was defined to have 100% ETP.
Plasma assays were performed by technicians unaware of the women’s trimester or characteristics.
Statistical analysis
Data were analyzed using software (SAS, Version 9.2 [2002-2008]; SAS Institute Inc, Cary, NC). Due to the skewed distribution, log-transformed values were used for analysis of ETP levels. Median (quartile) values are given for description, unless indicated otherwise. Box plots show the median and the quartiles with whiskers indicating 1st and 99th percentile. Analysis of variance (ANOVA) models were used to test for differences in the levels of ProCGlobal and ETP between women with and without anticoagulation. The within-factor trimester was considered as a repeated factor in the ANOVA models. Furthermore, differences between healthy women with and without factor V Leiden were evaluated using ANOVA models, including the repeated factor trimester. All P values are 2-sided and P values < .05 are considered statistically significant.
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