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Pregnancy; |
National Center for Biomedical and Engineering Science,3 National University of Ireland Galway, Galway, Ireland
Department of Obstetrics and Gynecology,4 National University of Ireland Galway, Clinical Science Institute, University College Hospital Galway, Galway, Ireland
ABSTRACT
As thrombin is proposed to be involved in stimulating myometrial contractility during labor and preterm labor, we aimed to investigate the expression of prothrombin (F7), the precursor of thrombin, its receptors, the protease-activated receptor (PAR) family (F2R, F2RL1, F2RL2, and F2RL3), and prothrombinase FGL2 in human myometrium during pregnancy and labor. Messenger RNA and protein were isolated from human pregnant laboring and nonlaboring myometrial tissue and from human primary myometrial smooth muscle cells. Semiquantitative RT-PCR, real-time fluorescence RT-PCR, Western blotting, and fluorescence microscopy were performed to determine the expression levels of F7, FGL2, F2R, F2RL1, F2RL2, and F2RL3 in the myometrial tissues and cells. The expression of mRNA and protein for these molecules is reported for the first time in human myometrium at term pregnancy, at labor, and in the nonpregnant state. Importantly, an increase in F2R and a significant increase in F2RL3 mRNA expression at labor were demonstrated. Statistically significant increases in F2R and F2RL3 protein expression was also detected in human myometrium at labor. Furthermore, FGL2 mRNA expression at labor, and FGL2 protein expression at term pregnancy and at labor was observed in this tissue for the first time. The expression of F7, FGL2, F2R, F2RL1, F2RL2, and F2RL3 in human myometrium reveals that all the machinery necessary for thrombin activation and cellular activity is present in the myometrium during pregnancy and labor. These data, in conjunction with the demonstrated increase in F2R and F2RL3 expression at labor, suggest a principal role for these molecules in the regulation of myometrial function at labor, including preterm labor.
labor, myometrium, parturition, pregnancy, protease activated receptors, prothrombin, uterus
Thrombin is a serine protease that is activated from the zymogen prothrombin at the final step of the blood coagulation cascade, where it then converts fibrinogen to fibrin [1]. In addition to its role in the coagulation cascade, thrombin plays an important role in cellular activation. It promotes smooth muscle proliferation, regulation of cytokine production, modulation of vascular and nonvascular muscle contraction, platelet activation, and regulation of blood vessel diameter [2–7]. Furthermore, thrombin is generated from prothrombin in tissues (without activation of the coagulation cascade) by a tissue prothrombinase, fibrinogenlike protein 2 (FGL2) [8].
Thrombin mediates its cellular effects by activating a family of transmembrane G-protein-coupled protease-activated receptors (PARs) [9]. Following cleavage of the PAR extracellular N-terminus by a protease that requires a serine residue in its active site, a new receptor N-terminus is generated which acts as an intramolecular ligand by folding back onto itself and triggering receptor activation. There are four major PAR subtypes (F2R, F2RL1, F2RL2, and F2RL3, also known as PARs 1, 2, 3, and 4), of which F2R, F2RL2, and F2RL3 are activated by thrombin, and F2RL1 is activated by trypsin. The PARs are expressed in a wide range of tissue and cell types, including platelets, vascular and nonvascular smooth muscle, fibroblasts, and endothelial cells [9].
Previous studies have identified thrombin to be the active component in blood that is responsible for enhanced myometrial contractions [10–12]. Intrauterine bleeding in the form of unexplained bleeding or placental abruption is clinically associated with spontaneous abortion, preterm labor, and labor at term [13]. The role of thrombin in this pathophysiologic condition is unknown. It has been demonstrated, however, that thrombin elicited a contractile response in human and rat myometrium in vitro [10, 14, 15] and in rat myometrium in vivo [12], and it has been suggested that F2R is the major receptor for the stimulatory effect of thrombin in this tissue [14, 15].
Prothrombin protein expression has been demonstrated in porcine uterine smooth muscle and rat myometrium; however, mRNA expression was not detected in either tissue [16, 17]. Moreover, prothrombin mRNA expression was evident within the rat placenta and endometrium, and it was therefore concluded that myometrial prothrombin is taken up from these tissues [17]. The expression of thrombin receptor F2R, F2RL1, F2RL2, and F2RL3 mRNA has been observed in nonpregnant rat myometrium, whereas F2R mRNA, with a 10-fold increase, and F2RL3 were also expressed in pregnant rat myometrium [15, 18, 19]. F2RL3 expression has been detected in pregnant human myometrium [20], whereas prothrombinase FGL2 mRNA expression has been observed in pregnant and nonpregnant human myometrium [21]. There is no information regarding the expression of prothrombin, F2R, F2RL1, or F2RL2 in human myometrium, during late pregnancy or labor. Similarly, F2RL3 or FGL2 expression has not been observed at human labor.
In this study, therefore, we investigated the expression profiles of prothrombin (F7), F2R, F2RL1, F2RL2, F2RL3, and FGL2 at term pregnancy, labor, and the nonpregnant state in human myometrium by RT-PCR, real-time fluorescence RT-PCR, Western blotting, and microscopy.
Patient Recruitment and Tissue Collection
Patient recruitment took place in the Department of Obstetrics and Gynecology, University College Hospital Galway (UCHG), Ireland. The study was approved by the Research Ethics committee of UCHG, and recruitment was carried out by provision of information sheets and obtaining written informed consent. Biopsies of myometrium were excised from the midline of the upper lip of the uterine incision made at cesarean delivery at elective (pregnant not in labor [PNL]) and intrapartum (pregnant in labor [PL]) procedures and at a preterm (PT) period of gestation. Reasons for emergency delivery included breech presentation, previous cesarean delivery, and abnormal fetal position. The criteria for inclusion in the intrapartum group were regular spontaneous uterine contractions, effacement of the cervix, and cervical dilatation greater than 3 cm prior to cesarean delivery. Samples of nonpregnant myometrium (NP) were excised from the body of the uterus of hysterectomy specimens. Women with malignant conditions and those receiving exogenous hormone therapy (e.g., progestagens) were excluded from the study. A sample of PT myometrium was obtained from cesarean delivery performed at 32 wk gestation.
Myometrial Cell Isolation and Culture
Myometrial tissue samples were digested in sterile, filtered Dulbecco modified Eagle medium (minus calf serum) containing 1 mg/ml collagenase type IA and 1 mg/ml collagenase type XI and 0.1% BSA (Sigma-Aldrich, Dublin, Ireland). After separation and washing, the cells were cultured in SGM-2 medium (Cambrex; Biowhittaker UK Ltd., Wokingham, UK) at 37°C and 5% CO2. Human myometrial smooth muscle cells subpassaged from primary cells in culture were characterized for mRNA expression of calponin and estrogen receptor-
and for alpha-smooth muscle actin mRNA and protein expression.
Tissue Samples for mRNA Expression
Biopsies of myometrium during pregnancy were obtained at the time of elective (n = 6) and intrapartum (n = 6) cesarean delivery. The reasons for elective cesarean delivery included previous cesarean delivery (n = 5) and placenta previa (n = 1). The reasons for emergency cesarean delivery were face presentation (n = 4), suspected fetal distress (n = 1), and previous classical cesarean delivery (n = 1). The mean age of the women was 34.83 yr (range, 29–41 yr); 2 were primagravida and 10 were multigravida. All women were delivered between 37 and 42 wk gestation.
Total RNA was isolated from frozen human myometrial tissue with TRIzol Reagent (Life Technologies, Paisley, UK) [22] and from the cells with the RNeasy RNA isolation kit (Qiagen, Crawley, UK). RNA was DNase treated with a DNA removal kit (Ambion, Huntingdon, UK).
Reverse Transcription-Polymerase Chain Reaction
Purified RNA samples (500 ng) were reverse transcribed using oligo (dT)15 primer and MMLV reverse transcriptase (Promega, Southampton Science Park, UK). Control RNA samples, in which no reverse transcriptase was added, were included.
A total of 0.5–1 µl of the 20-µl RT reaction amplified with BioTaq DNA polymerase (Bioline Ltd., London, UK). Primer pairs were designed where possible to flank predicted splice sites. The sequences of the oligonucleotide primers were:
ACTB: sense 5'-CAA CTC CAT CAT GAA GTG TGA C-3', antisense 5'- GCC ATG CCA ATC TCA TC-3', accession M10277; FGL2: sense 5'-ATT CAG CTT CTC CAG GCG ACC-3', antisense 5'-TTC CTT CCT TAA GCT TGC ACG ACT GTA AAG ATG AAG CTG-3' [23]; F7: sense 5'-TCC TCC ACG GCT ACG GAT GTG T-3', antisense 5'-TGC ATT CCT GCC TCC TCA CGG T-3', accession NM_000506; F2R: sense 5'-CTC GAA TTC TGA AGG TCA AGA AGC CGG-3', antisense 5'-CTC GAA TTC AGC TTT TTG TAT ATG CTG-3' [24]; F2RL1 sense 5'-AGA AGC CTT ATT GGT AAG GTT-3', antisense 5'-AAC ATC ATG ACA GGT CGT GAT-3' [25]; F2RL2: sense 5'-CTG ATA CCT GCC ATC TAC CTC C-3', antisense 5'-AGA AAA CTG TTG CCC ACA CC-3' [25]; and F2RL3: sense 5'-ATT ACT CGG ACC CGA GCC-3', antisense 5'-TGT AAG GCC CAC CCT TCT C-3' [25].
Polymerase chain reaction products were separated on agarose gels and visualized after ethidium bromide staining by UV illumination. Bands identified were purified with a Gel Extraction kit (Qiagen) and sequenced (MWG-Biotech, Milton-Keynes, UK).
Real-Time Fluorescence RT-PCR Using ABI 7700 Technology
Real-time PCR was performed on a 1:125 dilution of each of the six PNL and six PL myometrial cDNAs in triplicate for each transcript using the Applied Biosystems ABI Prism 7000 Sequence Detection System (ABI). Polymerase chain reaction was performed using SYBR Green PCR Master Mix (ABI), 5 µl diluted cDNA, and 0.4 µM of each primer. The amplification program was 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec, and 60°C and 72°C for 30 sec each. The sequences of the real-time fluorescence oligonucleotide primers were: F2R: sense 5'-CAA ATG CCA CCT TAG ATC CCC-3', antisense 5'-CTT CTG AGA TGA ATG CAG GAA GT-3' [26]; F2RL3: sense 5'-GCT GCT GCA TTA CTC GGA C-3', antisense 5'-ACG TAG GCA CCA TAG AGG TTG-3' [26]; and ACTB: sense 5'-GGG CAT GGG TCA GAA GGA TT-3', antisense 5'-AGT TGG TGA CGA TGC CGT G-3' (accession M10277).
Fluorescence data was acquired at the end of each PCR cycle. Melting curve analysis was performed by an initial denaturation step of 95°C for 15 sec, cooling to 60°C for 10 sec and, finally, 95°C for 15 sec.
The mean cycle threshold (Ct) of each gene for every patient from his or her standard curves (which were performed in triplicate) was normalized to the corresponding mean Ct value for the housekeeping gene, β-actin (ACTB). The normalized Ct values of the six PNL and the six PL myometrial tissue types (PNL vs. PL) were compared using the independent samples t-test, and then averaged. Results were expressed as mean normalized Ct units ± SEM. A P value of < 0.05 was considered to be statistically significant. Relative fold changes then were calculated using the difference in the mean normalized Ct values (x) between the pregnant at-term and the laboring myometrium for each transcript, relative fold change = 2x. All statistical analysis was performed using the SPSS statistical package (Statistical Package for the Social Sciences, v.14; SPSS Inc., Chicago, IL).
Tissue Samples for Protein Expression
Biopsies of myometrium during pregnancy were obtained at elective (n = 1) and intrapartum (n = 1) cesarean delivery at the time of hysterectomy (n = 1) and preterm labor (n = 1). The reason for the elective cesarean delivery was previous cesarean delivery (age 37 yr, multigravida). The reason for emergency cesarean delivery was fetal distress (age 33 yr, primigravida). The reason for hysterectomy included menorrhagia and fibroids. The age of the woman undergoing hysterectomy was 45 yr. One preterm sample was obtained by cesarean delivery at 32 wk from a woman age 29 yr who was primagravida.
Biopsies of myometrium during pregnancy were obtained for the FGL2 and the quantitative Western blot experiments at elective (n = 4) and intrapartum (n = 5) cesarean delivery. The reasons for elective cesarean delivery included polycystic ovary disease (n = 1) and previous cesarean delivery (n = 3). The reasons for emergency cesarean delivery were fetal distress (n = 3), failed induction (n = 1), and failure to progress (n = 1). The mean age of the women was 35.5 yr (range, 30–41 yr), of whom four were primagravida and five multigravida. All women were delivered between 39 and 40 wk gestation.
Human myometrial tissue was homogenized in 50 mM Tris, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, 10% glycerol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF (Sigma-Aldrich). Cellular debris was removed by centrifugation at 10 000 x g, 4°C for 5 min. The resultant supernatant was used for Western blot analysis.
Protein samples (30 µg) were resolved by electrophoresis on 7.5%–12% SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA) and electroblotted. Membranes were blocked with 1x PBS containing 0.05% Tween-20 (Sigma-Aldrich) and 5% lowfat milk powder (Dawn Dairies, Westmeath, Ireland). Blots were incubated with a 1:500 dilution of F7 (K-20 sc 16972) or 1:500 dilution of F2RL2 (C-20-sc8208) or 1:500 dilution of F2RL3 (C-20-sc8464) goat polyclonal IgG anti-human primary antibodies (Santa Cruz Biotechnology Inc., Heidelberg, Germany); with a 1:1000 dilution of F2R mouse polyclonal anti-human antibody (BD Transduction Laboratories, BD Biosciences, San Jose, CA); with 1:500 dilution of F2RL1 (SAM11-sc13504; Santa Cruz Biotechnology); with 1:1000 dilution of FGL2 (M01) clone 6D9 (Abnova Corp., Taipei, Taiwan) or 1000–10 000 dilution of β-actin (ACTB) clone number AC-15 (Sigma-Aldrich) mouse monoclonal IgG anti-human primary antibodies, diluted in 1x PBS containing 3% BSA and 0.03% Tween-20 or in 1x PBS with 5% lowfat milk powder (Dawn Dairies) with 0.03% Tween-20, for 1 h at room temperature or in 5% lowfat milk powder (Dawn Dairies) with 0.03% Tween-20 and 0.1% NaN3 overnight at 4°C, and for FGL2 (1x PBS, 0.2% Tween-20, 5% milk powder). After washing, blots were incubated in a 1:5000 dilution of a goat anti-mouse horseradish peroxidase (HRP)-conjugated antibody (sc2005; Santa Cruz Biotechnology) or with a 1:2000 to 1:4000 dilution of a rabbit anti-goat horseradish peroxidase-conjugated antibody (DakoCytomation Ltd.) in 1x PBS, 5% lowfat milk powder (Dawn Dairies) with 0.05% Tween-20 or in 1x PBS, 3% BSA, 0.05% Tween-20 for 1 h at room temperature. After washes in PBS 0.05%, the HRP-bound secondary antibody was detected using the Pierce West-Pico or the Super Signal West Dura chemiluminescent detection kits (Pierce Technology, Rockford, IL). The membranes were scanned with a digital imaging system (FluorchemTM 8900; Alpha Innotech Corp., San Leandro, CA) or were put on film (Nishiazabu 2-chome; FujiFilm, Tokyo, Japan). Protein expression levels were determined by densitometric analysis using the AlphaEaseFC software (Alpha Innotech). Protein expression levels were compared to ACTB levels. Densitometric values for F2R, F2RL3, and ACTB were determined, averaged, and expressed as normalized mean densitometric units ± SEM. Independent-sample t-tests were performed on the data using the statistical package SPSS for Windows version 14. A P value < 0.05 was considered to be statistically significant.
Human myometrial smooth muscle cells passaged from primary cells in culture (passage 7) were used for immunofluorescence microscopy. Cells were cultivated on 25-mm tissue culture coverslips (Sarstedt Inc.) in six-well dishes and fixed in 4% paraformaldehyde/2% sucrose solution and permeabilized in ice-cold 1x PBS 0.01% Triton X-100 for 5 min. Cells were then incubated with 1:100 dilution of each primary antibody (as above) or anti--smooth muscle actin mouse (ACTA2) monoclonal IgG anti-human antibody clone number 1A4 (Sigma-Aldrich) in PBS/1% BSA for 1 h at room temperature. Samples were incubated with an anti-goat IgG tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibody (Sigma-Aldrich) or an anti-mouse IgG fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Sigma-Aldrich). Control cells were incubated with the secondary antibody alone. After washing, the coverslips were mounted on glass slides with Fluormount mountant (VWR International Ltd., Poole, UK). Confocal images were obtained using the Laser Scanning Microscope LSM 510 (Carl Zeiss AG, Oberkocken, Germany).
RT-PCR analysis using DNA-free (Ambion) treated RNA demonstrated expression of F7, FGL2, F2R, F2RL2, and F2RL3 in nonpregnant, nonlaboring, and laboring human myometrium and F2RL1 in nonlaboring and laboring human myometrium (Fig. 1). The absence of transcripts in reverse transcriptase-negative reactions (RT–) confirmed that all products were RNA derived and not generated from contaminating genomic DNA.
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To determine cellular expression of the protease-activated receptors, RT-PCR analysis was also performed using DNA-free treated RNA from primary myometrial smooth muscle cells (passage 6) where F2R, F2RL1, F2RL2, and F2RL3 mRNA expression was detected (data not shown).
Quantitative real-time fluorescence RT-PCR was subsequently performed on F2R and F2RL3 mRNA from human myometrium as a result of slight increases in expression observed by semi-quantitative RT-PCR.
Relative quantitative expression analysis was performed by real-time RT-PCR on mRNA isolated from pregnant laboring and pregnant nonlaboring human myometrium tissue. In order to minimize any undue experimental error from sources such as pipetting inaccuracies, analyses of each gene were performed in triplicate. All nonlaboring and laboring myometrial biopsies demonstrated expression of F2R, F2RL3, and ACTB mRNA. RT-PCR product specificity was confirmed using melting curve analysis. Amplification curve crossing points were determined for each gene generated within the initial phase of exponential amplification per 0.5 µg total RNA in the tissues studied. ACTB expression showed no significant difference between the different tissue types. The mean (Ct) value of each gene for each patient (PCRs were performed in triplicate) at the different pregnancy stage was normalized to that of the corresponding mean ACTB Ct value, and the resultant values averaged.
These values, F2R mean normalized Ct values (per 0.5 µg total RNA) ± SEM were: PNL (n = 6) 34.02 ± 1.06715; and PL (n = 6) 30.855 ± 0.97774, P = 0.054. These data are graphically represented in Figure 2a. The relative fold-change was calculated from the difference in the mean normalized Ct values (x) between the pregnant laboring and nonlaboring myometrium, relative fold change = 2x (i.e., 34.02–30.855, 23.165). This resulted in a 9-fold upregulation of F2R expression at labor, although this was not statistically significant (P = 0.054).
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The F2RL3 mean normalized Ct values (per 0.5 µg total RNA) ± SEM were: PNL (n = 6) 38.4505 ± 1.03515; and PL (n = 6) 35.15 ± 0.64505, P = 0.022. These data are graphically represented in Figure 2b. A statistically significant 10-fold upregulation in F2RL3 mRNA expression at labor was calculated from the difference in the Ct values (x) between the pregnant laboring and nonlaboring myometrium (P = 0.022). A summary of the fold-changes in F2R and F2RL3 gene expression at labor as observed by real-time fluorescence RT-PCR is graphically represented in Figure 2c.
Western blotting demonstrated expression of F7, F2R, F2RL1, F2RL2, and F2RL3 protein in nonpregnant, nonlaboring, and laboring human myometrium, and FGL2 expression in nonlaboring and laboring myometrium (Fig. 3). F7, F2RL1, and F2RL2 expression was also observed in preterm labor (Fig. 3, a, d, and e). FGL2 expression did not alter significantly between the laboring and nonlaboring myometrial samples (Fig. 3b). The expression of F7, F2RL1, and F2RL2 protein did not alter significantly in the various pregnancy, labor, and nonpregnant states (Fig. 3, a, d, and e). F2RL3 expression increased during pregnancy and labor in comparison with the nonpregnant state (Fig. 3f). F2R and F2RL3 myometrial protein expression increased at labor in comparison with the nonlaboring state (Figs. 4 and 5). The ACTB protein levels did not appear to differ among the pregnant nonlaboring and pregnant laboring myometrial samples. Quantitative densitometric analysis was performed in which F2R and F2RL3 protein expression was compared to the corresponding ACTB protein expression on the same respective membranes, PNL (n = 3) and PL (n = 3; Figs. 4b and 5b). The averaged densitometric units (per 30 µg total protein) ± SEM for F2R were: PNL, 1863414.7 ± 26089.543; and PL, 2488044 ± 189312.21, and for the corresponding ACTB performed on the same protein gel membrane (per 30 µg total protein) were: PNL, 1072757 ± 91083.35; and PL, 915023 ± 27054.09. A graphic representation of this F2R quantitative data is presented in Figure 4b. Statistical analysis revealed a significant increase of 34% in F2R protein expression in human myometrial tissues at labor (P = 0.031). No significant difference in the corresponding ACTB protein expression was observed between the two pregnancy states.
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The averaged densitometric units (per 30 µg total protein) ± SEM for F2RL3 were: PNL, 5346733 ± 252821.2; and PL, 7970816 ± 851152.6, and for the corresponding ACTB performed on the same protein gel membrane (per 30 µg total protein) were: PNL, 1111465 ± 5378.8; and PL, 1199348 ± 42607.4. A graphic representation of this F2RL3 quantitative data is presented in Figure 5b. Statistical analysis revealed a significant increase of 50% in F2RL3 protein expression in human myometrial tissues at labor (P = 0.042). No significant difference in the corresponding ACTB protein expression was observed between the two pregnancy states.
Immunolabeling confocal microscopy was used to determine the localization of F7, F2R, F2RL1, F2RL2, and F2RL3 on human primary myometrial smooth muscle cells, passage 7 (Fig. 6). Prothrombin protein was localized to the cytoplasm. Both F2R and F2RL1 fluorescence localized to myometrial smooth muscle cell membrane and cytoplasm. F2RL2 localized to the nucleus and membrane, whereas F2RL3 protein was localized to the cytoplasm and cell membrane of the myometrial smooth muscle cells.
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In this study we have reported for the first time the expression of F7, F2R, F2RL1, and F2RL2 in the human myometrium, as well as the upregulation of F2R and F2RL3 mRNA and protein expression, in the myometrium during labor.
Previous studies have demonstrated prothrombin protein in the rat myometrium and suggested that presynthesized prothrombin is taken up from the placenta and endometrium [17]. Our investigation reveals, however, that prothrombin is expressed in human myometrium. Thrombin has been shown previously to stimulate contractility in both human and rat myometrium [12, 14, 15]; now, its inactive zymogen, prothrombin, is reported to be expressed in this human tissue. Confirmation of expression of prothrombin in human myometrium is a novel finding, and it suggests that it could possibly play an important role in the local regulation of myometrial contractility.
FGL2 mRNA expression had previously been observed in human myometrium [21]; however, this is the first report of its expression at labor and also of FGL2 protein expression at term pregnancy and labor in this tissue. Functional prothrombinase activity was also detected in pregnant and nonpregnant rat myometrium [27]. The presence of FGL2 prothrombinase suggests that localized activation of myometrial prothrombin is possible, thus enabling thrombin to mediate its effects on uterine contractility.
This study revealed F2R expression in human myometrium throughout pregnancy and labor, where it is localized to the smooth muscle cells. Other investigators observed a 10-fold increase in F2R mRNA in rat pregnancy [15]; however, this is the first report of an increase in F2R mRNA expression (9-fold) at human labor, which was just outside statistical significance, and a statistically significant increase in F2R protein expression. This implies an important role for F2R activation in myometrial contractility at labor. Furthermore, other studies have demonstrated that F2R-activating peptides elicited potent uterotonic effects in vitro, similar to those of thrombin, suggesting a major role for PAR-1 in thrombin-mediated myometrial contractility [14, 15].
No significant changes were observed in F2RL1 or F2RL2 myometrial expression at term pregnancy or labor, where expression was localized to the smooth muscle cells. F2RL3 expression was previously identified in human uterus [28], and F2RL3 mRNA expression was reported recently in pregnant and nonpregnant human myometrium [20]. In our investigation there was a significant increase in F2RL3 mRNA and protein expression at human labor, where its expression was localized to the myometrial smooth muscle cells.
In studies performed on human myometrium in vitro, an F2RL3-activating peptide exhibited an uterotonic effect, whereas an F2RL3 antagonist exhibited an uterorelaxant effect [20]. In the same study, thrombin exhibited a potent uterotonic effect, which did not, however, seem to be mediated by F2RL3, suggesting that F2RL3 may be activated by a protease other than thrombin. F2RL3 lacks a thrombin-binding, hiruidinlike sequence [29] and is known to mediate platelet activation by cathepsin G [30]. Therefore, in the human myometrium, F2RL3 may be activated by a protease other than thrombin. However, further investigation is necessary to elucidate the signaling pathways involved and the roles played by F2R and F2RL3 in the human myometrium at labor.
The increase in expression levels of F2R and F2RL3 in association with labor observed in this study suggest there could be upregulation of a thrombin-mediated uterotonic effect at this time. However, the exact signaling pathways linked to the uterotonic effect of thrombin are poorly understood, and they also may not be altered by the increased expression of F2R and F2RL3. This study did not examine this, and functional studies to investigate the mechanisms of action of thrombin and the possibility of increased sensitivity of the myometrium to it at labor will be an important topic of future study.
In summary, all of the machinery necessary for thrombin activation and cellular activity is present in the human myometrium, prothrombin, FGL2, and the PARs. The increase in expression of the PARs, F2R, and F2RL3 suggests a major role for these receptors in modulating human myometrial function at labor and preterm labor through thrombin and/or other proteases. Furthermore, thrombin and the PARs may also serve to regulate muscle tone after birth in the remodeling of the myometrium, which has to occur for it to return to its prepregnancy state.
ACKNOWLEDGMENTS
The authors are grateful to the medical and midwifery staff at University College Hospital Galway for their assistance with patient recruitment and tissue collection, and to the research nurse, Mary Quinn, for help with acquiring patient data. The authors acknowledge Elodie Ahamide for help with Western blotting and Deirdre O'Shaughnessy for help with protein isolation.
FOOTNOTES
1Supported by the Health Research Board of Ireland and the Higher Education Authority of Ireland's Program for Research in Third Level Institutions. 
Correspondence: 2Margaret O'Brien, National Center for Biomedical and Engineering Science, Orbsen Building, National University of Ireland Galway, University Road, Galway, Ireland. FAX: 353 91 492956; e-mail: margaret.obrien{at}nuigalway.ie
Received: 12 April 2007.
First decision: 4 May 2007.
Accepted: 14 September 2007.
REFERENCES
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60-kDa band.
