Objective
Normal pregnancy results in a prothrombotic state. Studies that have investigated the capacity of pregnant women to generate thrombin are limited. Our aim was to evaluate thrombin generation longitudinally from the preconception period, through pregnancy, and after pregnancy.
Study Design
We evaluated young, healthy nulligravid women (n = 20) at 4 time points and compared the data with 10 control women at 2 time points. Coagulation was initiated with tissue factor in contact pathway inhibited plasma, and thrombin generation was determined in the presence of a fluorogenic substrate.
Results
The maximum level and rate of thrombin generation increased during pregnancy; the highest level and rate occurred in late pregnancy compared with prepregnancy ( P < .001). Subsequently, thrombin generation decreased in the postpregnancy samples that included maximum level, rate, and area under the curve ( P < .001).
Conclusion
Our data provide evidence for an increase in tissue factor–dependent thrombin generation with pregnancy progression, followed by a return to prepregnancy thrombin levels.
Pregnancy presents a hematologic paradox. Despite hemorrhage being the leading cause of maternal death worldwide, pregnancy is a well-described hypercoagulable state, which confers significantly increased thrombotic risk. In more developed areas of the world, where hemorrhage is treated better and/or prevented, thromboembolic disease is the leading cause of maternal death. Indeed, pregnant women have a 4- to 5-fold increased risk of venous thromboembolism; the third trimester is the period of greatest risk. Pregnancy-related hormonal changes, particularly increases in estrogen levels, are thought to result in this shift to a more procoagulant state by resulting in increases in most clotting factors, a decrease in physiologic anticoagulants, and a decrease in fibrinolytic activity.
Empiric investigations into the mechanisms of enhanced coagulation in pregnancy are lacking. Most studies that have investigated the hemostatic system in pregnancy have described blood coagulation factors, fibrinolytic factors, and platelet function separately. More global tests of the hemostatic system, such as thrombin-generating capacity, have been used only recently. Thrombin generation captures the end result of a complex array of enzymatic reactions and interactions. Because of this, it has been hypothesized that the measurement of an individual’s capacity to generate thrombin, with the use of blood or plasma that has been subjected to a well-defined initiator, may be a better indicator of a thrombotic or hemorrhagic tendency than clot-based assays or comparative analyses of potential biomarkers. Therefore, the determination of tissue factor (Tf)–initiated thrombin generation during pregnancy is essential to a better understanding of the characteristic procoagulant state of normal pregnancy.
Data on thrombin generation in pregnancy are both limited and conflicting. There are few longitudinal studies that have evaluated thrombin generation over the course of pregnancy. Further, to our knowledge, there have been no studies performed that have evaluated the relationship between a woman’s capacity to generate thrombin outside of pregnancy and her ability to do so once pregnant. Likewise, and perhaps most importantly, there are no studies that have investigated whether pregnancy results in a persistent change in a woman’s capacity to generate thrombin. There have been multiple published studies that have suggested long-term, and even permanent, cardiovascular changes that have resulted from pregnancy. Whether pregnancy has a long-term effect on thrombin-generating capacity, however, is unknown.
In this study, we investigated a woman’s individual capacity to generate thrombin before pregnancy, in early and late pregnancy, and approximately 1 year after pregnancy (n = 20). Additionally, control studies (n = 10) were performed in nonpregnant women over a 2-3 year time period (average, 30 months). Plasma samples from each time point in all 30 women were evaluated for thrombin-generating capacity through the Tf pathway.
Materials and Methods
Subjects
Plasma samples that were used in this study were collected by Hale et al, as previously described. In addition to plasma samples, a large body of clinical, physiologic, laboratory, and birth outcome data was collected and analyzed by Hale et al and made available to us. Briefly, 60 nulligravid women who intended conception were enrolled through an open advertisement. All participants were young (18-40 years), healthy, and nonsmoking, with no history of hypertension, diabetes mellitus, autoimmune disease, or clotting or bleeding disorders.
Study procedures
Study participants were placed on sodium and total calorie balanced diets for 3 days before each blood draw and were asked to abstain from alcohol and caffeine for at least 24 hours. Additionally, women were asked to avoid the use of decongestants and nonsteroidal medications for at least 48 hours before the study. All women had regular menstrual cycles at the time of study enrollment. Thirty women conceived; however, 8 women conceived before baseline prepregnancy studies were performed; 1 participant had a first-trimester miscarriage, and 1 participant was lost to follow-up evaluation. The remaining 20 participants who comprised the primary study population all conceived singleton pregnancies, had complete prepregnancy assessments, and delivered full-term liveborn infants. Three of the pregnant study subjects went on to experience complicated hypertension; 2 of these 3 women met strict criteria for preeclampsia, as previously reported by Hale et al . All 4 time-point plasma samples were available for only 1 of these 3 women; 3 time-point samples were available for the other 2 women.
All prepregnancy assessments were performed during the follicular phase of the menstrual cycle (prepregnancy sample). Assessments during pregnancy were performed at 11-15 menstrual weeks (early pregnancy sample) and again in the third trimester at 30-34 weeks (late pregnancy sample). Ovulation detection and early pregnancy ultrasound assessments were used to calculate gestational age. Postpregnancy blood draws were performed between 6 months and 2 years after delivery, once breastfeeding had been discontinued (postpregnancy sample). As with the prepregnancy draws, the postpregnancy blood draws were performed in the follicular phase of the menstrual cycle. All 4 plasma samples were available for 14 women. The women who originally were enrolled in the study of Hale et al and who did not become pregnant were continued in the study as control subjects (control time 1). Most of these women (n = 27) had a second blood draw performed an average of 2.5 years after the initial blood draw (control time 2). For this study, 10 of these subjects served as the control population.
For all subjects, blood samples were obtained without the use of a tourniquet and after at least 30 minutes of supine rest. Citrated platelet poor plasma was separated immediately with centrifugation, and the samples were frozen at −80°C. The research protocols were approved by the University of Vermont Human Investigational Committees. All women provided written informed consent.
Materials
Recombinant Tf (Tf 1-242 ) was provided as a gift from Drs Shu Len Liu and Roger Lundblad (Baxter Healthcare Corporation, Deerfield, IL). Corn trypsin inhibitor was isolated from corn as described elsewhere. Phosphatidylcholine and phosphatidylserine were purchased from Avanti Polar Lipids, Inc (Alabaster, AL). Phospholipid vesicles phosphatidylcholine and phosphatidylserine were prepared as described, comprising 75% phosphatidylcholine and 25% phosphatidylserine, and were used to relipidate Tf. The fluorogenic substrate benzyloxycarbonyl-Gly-Gly-Arg-7-amido-4methylcoumarin•HCl was purchased from Bachem Americas Inc (Torrance, CA) and prepared as previously described.
Thrombin generation assay
Previously frozen citrated platelet poor plasma samples were thawed at 37°C in the presence of 0.1 mg/mL corn trypsin inhibitor to inhibit the contact activation pathway (eg, intrinsic pathway). The thawed samples were then incubated with Ca +2 (15 mmol/L) and the slow-reacting fluorogenic substrate benzyloxycarbonyl-Gly-Gly-Arg-7-amido-4methylcoumarin•HCl (416 μmol/L) for 3 minutes. Blood coagulation was initiated with 5 pmol/L relipidated Tf 20 μmol/L phospholipid vesicles.
Thrombin generation was monitored continuously in a Synergy 4 plate reader that is powered by Gen5 data analysis software (BioTek US, Winooski, VT). Experiments were done in duplicate or in triplicate, depending on the volume of plasma available to us. The final volume added to each well included 80 μL plasma, 20 μL substrate/Ca2+, and 20 μL Tf reagent. For each sample, Tf-dependent thrombin generation was evaluated with a thrombin-generation curve. Each curve was analyzed for the peak rate of thrombin formation (peak rate), the maximum level of thrombin that was generated (max level), and the total thrombin that was generated (area under the curve [AUC]).
Statistical analysis
Data are presented as the mean ± SD unless otherwise stated. Repeated measures analysis of variance was used to evaluate thrombin-generation parameters across 4 time points. Pearson correlation coefficients were examined to test for correlations between study subject laboratory/physiologic data and thrombin generation. A probability value of < .05 was considered significant.
Results
Clinical characteristics of subjects
Demographic data and clinical characteristics for the 20 study subjects and the 10 control subjects are given in Table 1 . As shown, baseline demographic and/or clinical characteristics were not significantly different. Additionally, as part of the primary study by Hale et al, a wide array of physiologic and laboratory data were collected before pregnancy, in early and late pregnancy, and after pregnancy. These data included measurements of hemoglobin, platelet count, fibrinogen, and D-dimer. As expected in normal pregnancy, average hemoglobin levels decreased from 12.0 ± 0.8 g/dL before pregnancy to 11.2 ± 1 g/dL by late pregnancy; platelet levels decreased to a lesser extent, from 225 ± 49 × 10 3 /μL to 212 ± 30 × 10 3 /μL. Fibrinogen levels increased from 219 ± 46 mg/dL before pregnancy to 449 ± 133 mg/dL in late pregnancy. Similarly, D-dimer levels increased from 0.26 ± 0.15 mg/L before pregnancy to 0.97 ± 0.4 mg/dL in late pregnancy. Overall, findings were in agreement with expected physiologic and laboratory changes that have been reported in normal pregnancy.
| Characteristic | Prepregnancy | P value | |
|---|---|---|---|
| Subjects, n = 20 | Control subjects, n = 10 | ||
| Age, y | 29.8 ± 3.2 | 29.4 ± 5.4 | .82 |
| Weight, kg | 65.2 ± 10 | 63.5 ± 5.0 | .62 |
| Body mass index, kg/m 2 | 23.3 ± 3.2 | 23.3 ± 2.4 | .97 |
| Cycle day, n | 8.3 ± 3.8 | 9.6 ± 3.1 | .39 |
| Hemoglobin, g/dL | 12.9 ± 0.8 | 12.7 ± 0.7 | .66 |
Thrombin generation
Figure 1 and Table 2 show Tf-initiated thrombin-generation parameters and include max level, peak rate, and AUC (analogous to endogenous thrombin potential). The results in Figure 1 and Table 2 span the prepregnancy to the postpregnancy period for all 20 subjects. Maximum level of thrombin generation, peak rate, and AUC all significantly increased from the prepregnancy state to the early pregnancy setting ( P ≤ .001). As pregnancy progressed, the maximum level and peak rate of thrombin generation continued to significantly increase. The AUC showed a trend toward increased thrombin generation in the late pregnancy sample vs the early pregnancy sample, but this was not a statistically significant increase. As can be seen in Figure 1 , in the postpregnancy samples, thrombin generation in all parameters that were measured returned to levels that were consistent with prepregnancy values. This decrease in thrombin generation to baseline values was significant ( P ≤ .001) when compared to both early-pregnancy and late-pregnancy thrombin generation.
| Thrombin parameter | Prepregnancy (n = 19) | Early pregnancy (n = 19) | Late pregnancy (n = 20) | After pregnancy (n = 16) | Control subjects | |
|---|---|---|---|---|---|---|
| Time 1 (n = 10) | Time 2 (n = 10) | |||||
| Peak rate (RFU/s) a | 35 ± 18 | 97 ± 53 | 146 ± 77 | 50 ± 45 | 68 ± 43 | 57 ± 40 |
| Max level (nmol/L) b | 81 ± 41 | 219 ± 117 | 336 ± 178 | 101 ± 81 | 159 ± 100 | 133 ± 93 |
| Area under the curve (nmol/L × min) | 1162 ± 446 | 2157 ± 466 | 2410 ± 543 | 1392 ± 718 | 1553 ± 567 | 1480 ± 653 |
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