An Microsoft excel document containing quantitative parameters from thrombin generation and fibrin formation experiments.

An Microsoft excel document containing quantitative parameters from thrombin generation and fibrin formation experiments.</p

Primary ECs have low, variable levels of estrogen receptor gene expression.

(A) RNA-seq data from aortic EC were mined for coagulation genes of interest. (B) Estrogen receptor gene expression shown on a linear scale. HUVEC and HDMVEC whole cell lysates were probed for (C) ERα and (D) ERβ protein, using MCF-7 cell lysates and recombinant ERα and ERβ as positive controls. Empty wells are marked with an x. Blot is representative of N = 3.</p

OC hormones do not enhance EC-supported fibrin formation.

HUVEC and HDMVEC were treated with vehicle (0.7% ethanol [veh]), 10 ng/mL TNF⍺, 1 nM EE, 100 nM drospirenone (DRS), or EE and drospirenone (E+D) for 24 hours before RNA was extracted. Transcripts encoding (A) integrin ɑv (ITGAV), (B) integrin β3 (ITGB3), (C) plasminogen activator inhibitor type I (SERPINE1), and (D) tissue plasminogen activator (PLAT) were measured by RT-qPCR and normalized to the untreated control (N = 3–12; Bars = mean + SEM). TNFɑ was compared to untreated cells, whereas EE and drospirenone treatments were compared to vehicle (*p(E) HUVEC and (F) HDMVEC was measured by turbidity in recalcified NPP (curves representative of N = 4–5). Fibrin formation parameters and statistical analyses are provided in S2 File.</p

Lentiviral-mediated overexpression of <i>ESR1</i> and <i>ESR2</i>.

(A) HUVEC and HDMVEC tolerance of lentiviral transduction was measured by fluorescence 72 hours after lenti-mCherry transduction. (B) ESR1 and (C) ESR2 expression was measured by RT-qPCR and normalized to lenti-mCherry samples (N = 4). Relative expression of (D) ERα and (E) ERβ protein in HUVEC and HDMVEC following lentiviral transduction was normalized using MCF-7 immuno-dot blot intensity (N = 3). Bars = mean + SEM; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

OC hormones do not enhance EC-supported thrombin generation.

HUVEC and HDMVEC were treated with vehicle (0.7% ethanol [veh]), 10 ng/mL TNF⍺, 1 nM EE, 100 nM drospirenone (DRS), or EE and drospirenone (E+D) for 24 hours before RNA was extracted. Transcripts encoding (A) tissue factor pathway inhibitor (TFPI), (B) thrombomodulin (THBD), and (C) tissue factor (F3) were measured by RT-qPCR and normalized to the untreated control (N = 3–12; Bars = mean + SEM; *pD-G) Thrombin generation was initiated in recalcified NPP by (D) HUVEC and (E) HDMVEC after 24 hours of TNF⍺, or EE and/or drospirenone. Curves are representative of N = 7 experiments performed in duplicate. Thrombin generation initiated by exogenous tissue factor and propagated by HUVEC (F) or HDMVEC (G) was measured in NPP (N = 3). Thrombin generation parameters and statistical analyses are provided in S2 File.</p

Combined inflammation and overexpression of <i>ESR1</i> and <i>ESR2</i> in HUVEC and HDMVEC does not facilitate a prothrombotic response to EE.

Transcript expression of (A) TFPI, (B) THBD, (C) F3, (D) ITGAV, (E) ITGB3, (F) SERPINE1, and (G) PLAT was measured by RT-qPCR following 72 hours lentiviral transduction and 24 hours of subsequent 1 nM EE and/or 10 ng/mL TNFɑ treatment (N = 4; Bars = mean + SEM; *pFig 5 for comparison. Thrombin generation in NPP stimulated by (H) HUVEC or (I) HDMVEC treated with lentiviral vector and EE (single curves representative of N = 4 experiments in duplicate). Thrombin generation parameters and statistical analyses are provided in S2 File.</p

Contains supporting figures and tables.

BackgroundOral contraceptive (OC) use increases venous thromboembolism risk 2-5-fold. Procoagulant changes can be detected in plasma from OC users even without thrombosis, but cellular mechanisms that provoke thrombosis have not been identified. Endothelial cell (EC) dysfunction is thought to initiate venous thromboembolism. It is unknown whether OC hormones provoke aberrant procoagulant activity in ECs.ObjectiveCharacterize the effect of high-risk OC hormones (ethinyl estradiol [EE] and drospirenone) on EC procoagulant activity and the potential interplay with nuclear estrogen receptors ERα and ERβ and inflammatory processes.MethodsHuman umbilical vein and dermal microvascular ECs (HUVEC and HDMVEC, respectively) were treated with EE and/or drospirenone. Genes encoding the estrogen receptors ERα and ERβ (ESR1 and ESR2, respectively) were overexpressed in HUVEC and HDMVEC via lentiviral vectors. EC gene expression was assessed by RT-qPCR. The ability of ECs to support thrombin generation and fibrin formation was measured by calibrated automated thrombography and spectrophotometry, respectively.ResultsNeither EE nor drospirenone, alone or together, changed expression of genes encoding anti- or procoagulant proteins (TFPI, THBD, F3), integrins (ITGAV, ITGB3), or fibrinolytic mediators (SERPINE1, PLAT). EE and/or drospirenone did not increase EC-supported thrombin generation or fibrin formation, either. Our analyses indicated a subset of individuals express ESR1 and ESR2 transcripts in human aortic ECs. However, overexpression of ESR1 and/or ESR2 in HUVEC and HDMVEC did not facilitate the ability of OC-treated ECs to support procoagulant activity, even in the presence of a pro-inflammatory stimulus.ConclusionsThe OC hormones EE and drospirenone do not directly enhance thrombin generation potential of primary ECs in vitro.</div

Holds the raw dot blot images underlying Figs 3 and 4.

Holds the raw dot blot images underlying Figs 3 and 4.</p

Overexpression of <i>ESR1</i> and <i>ESR2</i> in HUVEC and HDMVEC does not facilitate a prothrombotic response to EE.

Transcript expression of (A) TFPI, (B) THBD, (C) F3, (D) ITGAV, (E) ITGB3, (F) SERPINE1, and (G) PLAT was measured by RT-qPCR following 72 hours lentiviral transduction and 24 hours of subsequent EE treatment (N = 4; Bars = mean + SEM; *p(H) HUVEC or (I) HDMVEC treated with lentiviral vector and EE (single curves representative of N = 4 experiments in duplicate). Thrombin generation parameters and statistical analyses are provided in S2 File.</p