Toward Prediction of Thrombosis in Adolescent Girls On Estrogen Containing Contraceptives: Global Coagulation Profiles and Estrogen Gene Polymorphisms.
Authors: Zia Ayesha N. , Callaghan Michael U , Sawni Anju , Backos Alcesa , Rao Latha , Chitlur Meera B. , Lusher Jeanne M. , Rajpurkar Madhvi 
Published: October 2019

Abstract
Abstract 2254

Background:
Adolescent girls on oral contraceptive pills (OCPs) are at a higher risk for venous thromboembolism. OCP induced changes on coagulation and fibrinolytic pathways are complex with high inter-individual variability and factor assays are often non- informative. Global coagulation assays better delineate OCP induced coagulation changes and variability in such changes may be the result of differences in estrogen metabolism.

Aims and Methods:
Our study aims to: 1) Determine the prevalence of a hypercoagulable state after initiation of OCPs as measured by global coagulation assays and 2) Determine the contribution of variation in estrogen metabolism to this hypercoagulable state. In this ongoing prospective study, 36 healthy non-pregnant adolescent girls starting OCPs were enrolled after institutional review board approval. Samples were obtained prior to and after 3 months of initiating OCPs and were analyzed for tests such as prothrombin time (PT), activated partial thromboplastin time (APTT), clotting and anticoagulant factor assays, tissue factor initiated thromboelastography (TF-TEG), with t-PA (tissue plasminogen activator) modification for detecting fibrinolysis (tpa-TEG), thrombin generation +/−thrombomodulin (TM)(TG+/− TM) and estrone levels. All participants were genotyped for FV Leiden, F2 G20210, plasminogen activator inhibitor-1 (PAI-1) promoter polymorphism and 3 single nucleotide polymorphisms (SNPs) related to estrogen metabolism (estrogen receptor a (ESRa), UGT1A1, CYP3A5). SNP genotypes were compared based on coagulation tests, TG and TEG parameters and differences pre and on OCPs were examined.

Results:

Baseline:
Median age of participants was 17 years (range 13–20). All were African American. Pre-OCP, the median FVIII activity was 160% (range 97–327) with normal values for other parameters.

On OCPs:
Routine tests:
There were no significant differences in blood counts, von Willebrand factor (vWF) antigen, FVIII, vWF, protein C, S, Anti-thrombin, or PAI-1 activity on OCPs. The PT was shorter (10.5 ± 0.4 sec vs. 11 ± 0.4 sec, p= <0.001) on OCPs, and there were no differences in APTT.
Thromboelastography:
TF-TEG demonstrated higher maximum amplitude (67 ± 5.4 mm vs. 72 ± 7 mm, p= 0.024)) and G(10 ± 2.4 vs. 13± 4.3 dynes/cm, p=0.021) on OCPs. There were no differences in time to clot initiation or the rate of clot formation. Tpa-TEG did not reveal any differences in clot lysis at 30 and 60 minutes on OCPs (p= 0.8 and 0.4 respectively).
Thrombin generation:
TG using calibrated automated thrombography in corn trypsin inhibitor treated plasma demonstrated a shorter lag time [without TM: 2.3 min ± 0.4 vs. 1.8 ± 0.3min, p= 0.001, with TM: 2 ± 0.33min vs.1.7 ± 0.32 min, p=0.005], higher endogenous thrombin potential [without TM: 921± 289 nM.min vs. 1145 ± 350 nM.min, p=0.08, with TM: 659 ± 222 nM.min vs. 945 ± 368 nM.min, p=0.029], higher peak [without TM: 228 ± 53 nm vs. 291± 68 nm, p=0.012, with TM: 202 ± 58 nm vs. 268 ± 70 nm, p=0.023) and shorter time to peak(without TM: 4.7 ± 0.7 min vs. 3.7 ± 0.5 min, p= 0.001, with TM: 3.9 ± 0.4 min vs. 3.4 ± 0.4 min, p= 0.007)after being on OCPs for 3 months. Non-group O blood type and elevated FVIII at baseline did not correlate with ETP with or without TM after being on OCPs. Estrone levels did not correlate with peak thrombin generation pre-OCPs (R= 0.1, p=0.8).
SNP genotyping:
The ESRa (c.454–397T>C) polymorphism was present in the heterozygous state in 51% and homozygous state in 15% of the participants respectively. CT and CC genotype was associated with shorter lag time with TG-TM (2.7 ± 0.25 min vs. 1.7 ±0.14 min, p=0.026) but not with TF-TEG. The ESRa, UGT1A1 and CYP3A5 polymorphisms did not correlate with estrone levels or other coagulation parameters. As anticipated, FVL was absent in all and F2 G20210 (heterozygous) was present in one participant.

Conclusions:
In vitro thrombin generation and clot kinetics measured by global coagulation assays reveal a state of hypercoagulability in adolescent girls on OCPs not detected by routine coagulation testing. These differences appear to be likely caused by acquired activated protein C resistance as the addition of TM had less effect in patients on OCPs despite similar factor VIII activity. The presence of CT and CC genotype of the 454–397T>C polymorphism in the estrogen receptor-a, independent of estrone levels, correlated with hypercoagulability based on TG parameters but not TF-TEG.

Disclosures:
No relevant conflicts of interest to declare.