Term Decidual Cell Thrombomodulin and Endothelial Protein C Receptor Expression Is Upregulated by Thrombin Irrespective of Ovarian Hormonal Priming.
Authors: Paidas Michael J. , Ku De-Hui W. , Arkel Yale S. , Tang Caroline , Fortunato Christine , Lockwood Charles J. 
Published: October 2019

Abstract
It is well known that normal pregnancy is associated with changes in the hemostatic system that favors a prothrombotic milieu. Moreover, pregnancies complicated by adverse pregnancy outcomes (APO), including fetal loss, intrauterine growth restriction, preeclampsia and abruptio placentae frequently have evidence of uteroplacental thrombosis, vascular damage, and fibrin deposition, upon pathologic examination. The protein C (PC) system [includes; PC, protein S (PS), thrombomodulin (TM), endothelial protein C receptor (EPCR)] is pivotal in the maintenance of the hemostatic balance. TM and EPCR are both transmembrane glycoproteins and are central to the generation of APC, down-regulation of thrombin (FIIa) formation, and down-regulation of the pro-inflammatory process. In trophoblasts TM EPCR are involved in regulating fibrin formation, trophoblast proliferation and apoptosis. Decidual cells (DCs) are located at the maternal fetal interface, an area that must undergo substantial remodeling of the extracellular matrix. We hypothesized that the DCs would express components of the PC system to regulate thrombin since it is involved in extracellular matrix degradation. DCs were harvested and prepared from term placentas from Cesarean sections from 5 uncomplicated pregnancies. The amnion was removed, and the decidua was scraped from the chorion, and digested. The DCs were purified using a Percoll Gradient, seeded on gelatin coated dishes, and passaged until free of white cells. Confluent DCs were incubated with vehicle control or primed in serum-containing medium with 10–8 M estradiol(E2), or 10–7 M medroxy-progesterone acetate(MPA), or E2+MPA. DCs were switched to defined medium with corresponding control and steroids with or without FIIa. After 6 hours, real time PCR was performed on extracted RNA. In the hormonally unprimed condition, thrombin tended to increase the expression of TM an average of 5 fold increase (n=5). The combination of E2+MPA revealed an average increase in the expression of TM in response to FIIa of 5.24 fold. This effect was neutralized by the addition of hirudin. In the hormonally unprimed condition, FIIa increased EPCR expression in response to FIIa, average of 2.18 (n=5). There was a slight further increase with the addition of FIIa 2.7, 2.1, 2.5 fold in the DCs primed with E2, MPA, E2+MPA, respectively. The effect was neutralized by the addition of hirudin. A western blot analysis of TM protein expression, a pattern similar to real time PCR was observed. The lack of differential response of FIIa to hormonal priming may be protective, particularly in the peripartum period, where there are significant hormonal fluctuations, yet FIIas regulatory mechanisms must be preserved. Further studies are underway to assess the interaction of the PC system, FIIa and the effect of gestational age on DCs. Derangements of PC system and DCs may predispose to APO.
Table 1
N=5 No stimulant T T + H No Stimulant T T+H Real time PCR normalized with beta-actin. T=thrombin (2.5U/ml), H=Hirudin (3U/ml). Column 2 to 4 are DCs without sterois priming. Column 5 to 7 are DCs primed with E2 + MPA. TM 1 +/− 0 5 +/− 5.24 1.08 +/− 0.43 3.08 +/− 1.49 5.24 +/− 2.28 1.85 +/− 2.2 EPCR 1 +/− 0 2.18 +/− 1.38 1.02 +/− 0.17 2.96 +/− 1.90 2.48 +/− 0.77 2.45 +/− 2.22