Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) reveals platelet activation in preeclampsia

Abstract Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) is a membrane protein receptor found in α-granules of megakaryocytes and platelets. Upon platelet activation TLT-1 is rapidly relocated to the surface of platelets. In plasma, a soluble form of TLT-1 (sTLT-1) is present. Plasma levels of sTLT-1 are significantly elevated in thrombotic diseases. In the present study, we investigated to whether TLT-1 reflects platelet activation in pregnant women with preeclampsia. We studied 30 preeclamptic patients who were matched with 30 normotensive pregnant women and 30 non-pregnant controls. Basal TLT-1, P-selectin, and CD63 expressions on platelets were analyzed with the use of flow-cytometry (FCM). Platelet reactivity was induced by thrombin receptor activation peptide and determined by FCM. Plasma concentrations of sTLT-1 and soluble P-selectin (sP-selectin) were measured by an enzyme-linked immunosorbent assay. Results show that basal platelet expression of TLT-1, P-selectin and CD63 were increased in women with preeclampsia (PE) compared with normotensive pregnant women (NP). Platelets from PE women and NP women were more responsive compared to from nonpregnant women controls (NC), and which was demonstrated by increased expression of TLT-1, P-selectin, and CD63 upon stimulation in vitro. Plasma concentration of sTLT-1 was greater in PE women compared to NP women and NC women. Plasma sP-selectin level was higher in pregnant women than in nonpregnant women, but there were no significant differences between PE and NP women. In summary, our results revealed that platelet activation is prominent in preeclampsia, TLT-1 reflects platelet activation and may be a useful indicator for preeclampsia.


Introduction
Preeclampsia is a serious pregnancy-related complication for both mother and fetus [1] and occurs in 3-5% of all pregnancies [2]. Currently, the diagnosis endorsed by the International Society for the Study of Hypertension in Pregnancy (ISSHP) embraces new onset hypertension (systolic >140 mmHg or diastolic >90 mmHg) accompanied by one or more other features: proteinuria, other maternal organ dysfunction (including liver, kidney, neurological), or hematological involvement, and/or uteroplacental dysfunction, such as fetal growth restriction and/or abnormal Doppler ultrasound findings of utero-placental blood flow [3].
The pathogenesis of preeclampsia is not fully elucidated but tremendous progress has been made over the past decade. The placenta has always been a critical figure in the etiology of preeclampsia because the removal of the placenta is necessary for symptoms to regress [4]. The abnormal spiral artery remodeling has been documented to be the central pathogenic factor in pregnancies complicated by intrauterine growth restriction, gestational hypertension, and preeclampsia [5]. Placental endotheliopathy is the root cause of preeclampsia and disseminates systemically to the maternal circulation. The shift has been made to view preeclampsia as a systemic disease with widespread endothelial damage and the potential to affect future cardiovascular diseases rather than a self-limited occurrence [6].
A protective hypercoagulable state is often developed during late pregnancy and can evolve into a prothrombotic state in patients with preeclampsia [7,8]. Additionally, increasing evidence showed platelet activation plays a vital role in the pathogenesis of preeclampsia [9][10][11]. Therefore, markers of platelet activation may have a potential for early prediction of preeclampsia.
Presently, flow cytometry (FCM) is the most sensitive technique to detect increased surface exposure of activation antigens on the platelet surface [12][13][14]. Activated platelets have a different exposure of glycoproteins on the surface of platelets that are measurable by flow cytometry. Platelet degranulation occurs with fusion of platelet granules and lysosomal membranes with the plasma membranes, which could be detected via surface expression of P-selectin (CD62P) [15] and CD63 [16], respectively. The expression of P-selectin and CD63 significantly increased in preeclampsia than that in non-preeclampsia [10,[17][18][19][20]. However, in clinical practice, these factors predicted just 30% of women who developed preeclampsia [21]. The test accuracy of these markers restrained the apply in clinical practice [22].
Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) is a membrane protein receptor specific to αgranules of megakaryocytes and platelets. Upon platelet activation TLT-1 is rapidly relocated to the surface of platelets [23,24]. TLT-1 is abundantly present in platelets, with expression levels greater than P-selectin in human (TLT-1,14200 copies; P-selectin, 8900 copies) [25]. TLT-1 facilitates platelet aggregation and mediates platelet interactions with both endothelial cells and neutrophils [26]. Smith et al. reported that surface expression of TLT-1 was a more sensitive marker of platelet activation than P-selectin [27]. A soluble form of TLT-1 (sTLT-1) also exists, the majority of sTLT-1 is shed from the surface of activated platelets, and the remainder is an alternatively splicing isoform stored and released from α-granules [28]. During inflammation, sTLT-1 may mediate hemostasis by enhancing actin polymerization, resulting in increased platelet aggregation and adherence to the endothelium [29]. Plasma levels of sTLT-1 are significantly elevated in sepsis [30] and coronary artery diseases [31]. Despite the proof of the biological functions TLT-1 plays on platelets and sTLT-1 plays in the plasma, there is little information about their role in preeclampsia.
Description of the status of platelets in preeclampsia, the determination of platelet properties (including quantification of basal [16] receptor levels and markers of ongoing activation), and platelet response to a standardized stimulus in vitro is another area of concern.
The primary purpose of our study was to investigate whether TLT-1 reflects platelet activation in pregnant women with preeclampsia. We inspected the TLT-1 expression on platelets in preeclamptic patients, healthy normotensive pregnant women and nonpregnant controls and the plasma concentration of sTLT-1 in these individuals. The sensitive markers of platelet activation, such as P-selectin and CD63, which have been closely linked to the TLT-1 were also studied. We further characterized the reactivity of platelets in the three groups stimulated with thrombin receptor activation peptide-6 (TRAP-6) in vitro.

Study Population
Pregnant women were recruited from the obstetrics and gynecology department of The First Affiliated Hospital of Soochow University from May 2020 to January 2021. For this study, we enrolled consecutive women with preeclampsia. Diagnostic criteria for preeclampsia were based on the American College of Obstetricians and Gynecologists Practice Bulletin Number 202 [32] and the Chinese Medical Association of Obstetrics and Gynecology Guidelines for hypertension in pregnancy (2020) [33]. Exclusion criteria were as follows: (1) usage of anticoagulant or antiplatelet drugs before enrollment; (2) history of hypertension, diabetes, chronic nephropathy, hepatitis, tumors, immune system diseases, and cardiac surgery before pregnancy; (3) history of thrombosis or bleeding disorders; (4) history of recurrent miscarriage; (5) in vitro fertilization and embryo transfer. Thirty preeclamptic patients (PE), thirty healthy normotensive pregnant women (NP) and thirty healthy age-matched nonpregnant controls (NC) were recruited for the case-control study. The normotensive pregnant women attended for routine antenatal care. They were matched for age and gestational age with the preeclamptic women. All pregnant women were followed-up until delivery. Both normotensive pregnant and nonpregnant women denied taking any medication in the previous 2 weeks. The study was approved by the local ethics committee. All participants gave written informed consent.

Blood Sampling
Two fasting blood samples were drawn from the antecubital vein without tourniquet through a 21-gauge needle with a vacutainer system. The first sample was collected in a 2.0 mL ethylenediaminetetraacetic acid (EDTA) K 2 tube (BD, NE, USA) for FCM assay. The second sample was collected into a 2.7 mL tube containing 0.105 M buffered sodium citrate (BD, Devon, UK). Within 30 minutes after collection, cells were removed by centrifugation for 10 minutes at 1500 g and 20°C. Plasma samples were then divided in 200 uL aliquots, immediately snap frozen in liquid nitrogen and stored at −80°C for further assay. The samples were performed assay within 2 hours of blood collection. All laboratory tests were performed blinded to as case or control status.

Flow Cytometry Assay
Whole blood flow cytometric analysis of clinical samples were modified from Janes et al. [34]. Fifty μL EDTA-anticoagulated whole blood was diluted with 450 μL modified tyrodes's buffer (2.5 mM HEPES, 150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO 3 , 5.5 mM D-glucose, 1 mM CaCl 2 , 1 mM MgCl 2 , pH 7.4). When studying reactivity of platelets in vitro, 49 μL of diluted whole blood were incubated with 1 μL TRAP-6 for 20 minutes at 37°C [10], to reach final concentration of 5 μM. In parallel, same amount of whole blood was incubated with modified tyrodes's buffer as basal status. The mixed suspensions were incubated with FITC-or PE-conjugated antibody for another 20 minutes at room temperature in dark and then mixed with 500 μL of 0.2% (v/v) PFA-PBS (paraformaldehyde (PFA) in phosphate-buffered saline (PBS)) to inhibit further activation, and analyzed through Flow cytometry (FC500, Beckman Coulter) with CXP Software Version 2.3 within 2 hours of collection.
The platelet population was identified by light-scatter characteristics and enclosed in an electronic bit-map. Daily control of fluorescence intensity was done with Flow-Set fluorescence microspheres (Coulter Corporation, Miami, FL) according to the manufacturer's instructions. Five thousand platelets were analyzed from each sample. Results were expressed as percentage of positive platelets, which defines the negative control (isotype control) to 0%. Also, mean fluorescence intensities (MFI) were presented, which were calculated for gated cells. Samples from normotensive pregnant women and nonpregnant women were run in parallel with those from preeclampsia patients.

Enzyme-Linked Immunosorbent Assay
All plasma analytes were subjected to ELISA assay to determine the plasma concentration of sTLT-1 and sP-selectin. Quantitative analysis of plasma sTLT-1 was performed as the manufacturer's instructions. Briefly, 100 μL of plasma samples was added to a pre-coated 96 well plate and incubated for 2 hours at 37°C. After 2 hours, the solution was removed without washing. 100 μL of biotinylated anti-TLT-1 antibody was added and incubated for 1 h at 37°C. After 1 h, the solution was discarded and the plate was washed 3 times with 1× washing. After the last wash, removed any remaining washing buffer by inverting the plate and blotting against a paper towel. Then, 100 μL of horse radish peroxidase (HRP)-conjugated streptavidin was pipetted to the wells for another 1 h at 37°C. Repeated wash was performed as above. Then, 100 μL of TMB one-step substrate reagent was added and incubated for 15 minutes at 37°C in the dark. After 15 minutes, 50 μL of stop solution was added and the plate was read at 450 nm using a microplate reader (SpectraMax M2, Molecular devices) with SoftMax Pro software, four-parameter fitting curve was drawn through the standard points. The exact concentration of sP-selectin (diluted 1:50) was detected by ELISA, according to the manufacturer's instructions. The sensitivity of sTLT-1 assay is 31.25 pg/ml, and inter-and intra-assay coefficients variation (CV) is 12.35% and 4.78%. The sensitivity of sP-selectin assay is 20 pg/mL, and inter-and intra-assay CV is 8.24% and 6.53%.

Statistical Analyses
All data were analyzed with Graph Pad Software version 7.0. The normality of the data was calculated using the D'Agostino & Pearson omnibus normality test. Data were presented as mean with standard deviation (SD) or as median with inter-quartile range (IQR) for normally distributed data and skewed data, respectively. For normally distributed data, one-way ANOVA analysis was used to compare the multiple groups; intergroup comparisons were performed using Tukey's post-hoc test. Student's t test was conducted to compare two groups. For skewed data, nonparametric tests Mann-Whitney U were used to compare the difference among groups. P < .05 was considered statistically significant.

Clinical Characteristics
The Clinical data are depicted in Table I. The three groups were of comparable age, and the pregnant women were of comparable gravidity and parity. As expected, compared to normotensive pregnant women (NP), preeclamptic women (PE) had significantly higher body mass index, systolic and diastolic blood pressure, as well as lower gestational age (P < .01). Pregnant women had significantly lower platelet counts than that in nonpregnant women controls (NC) (P < .01), while there was no significant difference between the preeclamptic and normotensive pregnant women. The median duration of the pregnancy at delivery was 4 weeks shorter for the preeclampsia group than for the NP group (P < .01) as would be expected. Infants delivered by women with preeclampsia had significantly lower birth weights when compared to health pregnancy group (P < .001).

Basal Platelet Activation Status
Data related to platelet basal expression of TLT-1, P-selectin and CD63 are presented in Figure 1. TLT-1 expressed on platelet surface in women with preeclampsia was significantly higher when compared to in NP group and in NC group, which was demonstrated both as the percentage of positive platelets and as MFI levels. TLT-1 expression implied both as the percentage of positive platelets and as MFI levels in NP women was significantly higher than in NC women.
Similar as TLT-1, compared with the nonpregnant women, P-selectin expressed on platelet surface in pregnant patients including preeclampsia women and NP women elevated significantly. The expression of P-selectin in PE women was higher than in NP women indicated as the percentage of positive platelets.
The expression of CD63 upregulated significantly in women with preeclampsia when compared with in NP women and in NC women, which implied as the percentage of positive platelets. The MFI levels of CD63 in pregnancy women (preeclampsia women and NP women) were significantly higher than in NC women, while there was no difference between in PE and in NP women. The detailed data and statistical conclusion of basal platelet activation status were summarized in supplementary information 1.

In Vitro Platelet Reactivity
Data related to platelet reactivity in vitro are summarized in Table  II. On stimulation with 5 μM TRAP-6 in vitro, the median of TLT-1 positive platelets percentage in women with preeclampsia and in NP women was 62.88% and 64.92%, respectively, which were significant higher than in NC women (44.42%). However, there was no significant difference between in PE women and in NP women. The platelets surface expression of TLT-1 showed as MFI levels in PE women were significantly higher when compared with in NP women and in NC women. P-selectin expression in pregnant women (preeclampsia women and NP women) was significantly higher than in nonpregnant women, which was indicated as the percentage of positive platelets. There was no significant difference between in PE patients and in NP women.
With regard to CD63 expression, indicated as the percentage of positive platelets, in women with preeclampsia showed significantly higher platelet reactivity than in NC women, while there was no difference between in PE and in NP women.

Soluble TLT-1 and P-selectin in Plasma
Individual values of plasma soluble TLT-1 and P-selectin levels are shown in Figure 2. The median concentration of sTLT-1 in the preeclamptic group was 78.95 pg/ml, which significantly higher than in NP women (44.25 pg/ml) and in nonpregnant controls (47.23 pg/ml). There was no significant difference between in NP women and in NC women. The median concentration of sPselectin in PE women was 87.58 ng/ml, which was significantly higher than in NP women (68.51 ng/ml) and in NC women (47.22 ng/ml). The median concentration of sP-selectin in NP women was significantly higher than in NC women (P < .01). The detailed data and statistical conclusion of soluble TLT-1 and P-selectin plasma concentration were summarized in supplementary information 2.

Discussion
To our knowledge, this is the first report of a significantly enhanced platelet surface expression and plasma levels of TLT-1 in preeclamptic patients. Compared with those of normotensive pregnant women and nonpregnant women controls, TLT-1 positive platelets percentage levels and plasma sTLT-1 in preeclamptic patients were markedly increased. Our study demonstrated that TLT-1 reveals platelet activation in preeclampsia, which was demonstrated as both higher TLT-1 positive platelet percent and elevated plasma sTLT-1 in preeclamptic patients when compared with normotensive pregnant women and nonpregnant women controls. Therefore, the elevated expression level of TLT-1 is a novel finding in preeclampsia and essentially supports the concept of platelet activation in this disease. Platelets membrane glycoprotein, expressed on platelets and/or soluble forms of which shed from platelets have been identified as markers for platelet activity in vivo [15,35,36]. As such, TLT-1 joins the growing list of markers of platelet hyperactivity described in this disorder. Preeclampsia (PE), the leading cause of maternal and fetal morbidity and mortality, is associated with poor fetal growth, intrauterine growth restriction (IUGR) and low birth weight (LBW) [37]. The placenta plays a vital role in the etiology of PE in the mother and her child. PE alters normal placental development during pregnancy resulting in short-and long-term complications for both the mother and the offspring. PE is a leading contributor to IUGR and numerous preclinical models that mimic the pathogenesis of PE demonstrate that IUGR offspring exhibit sex-and age-specific increases in blood pressure and cardiovascular risk [38]. In our finite cohort of 30 preeclamptic women, 16 cases of LBW were reported, during them 6 cases complicated with IUGR. P-selectin, an adhesion molecule in the secretory granules of platelets, is a sensitive and specific index of platelet activation. Circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, and hence platelet surface P-selectin is not an ideal marker for the detection of circulating degranulated platelets [39]. P-selectin also presents in Weibel-Palade bodies of endothelial cells [35]. This implies that sP-selectin in plasma can originate from endothelial cells and/or platelets. However, strong correlations between plasma sP-selectin concentration and platelet count as well as α-thromboglobulin [9], and a lack of correlation with endothelial activation markers [11], suggest that plasma sP-selectin most likely originates from platelets. Therefore, soluble P-selectin reflects the continuous activation status of platelets in vivo, may still be a useful marker of platelet activation in clinical settings. Multiple reports have shown significantly enhanced platelet surface expression and plasma levels of sPselectin in women with preeclampsia compared with normotensive pregnant women [10,11]. Our study found significantly enhanced platelet surface expression and plasma levels of sPselectin in women with preeclampsia compared with normotensive pregnant. We also found the higher basal P-selectin levels and being more responsive to in vitro agonist stimulations in women with preeclampsia compared with NP women. These results are in keeping with findings of other investigators [10,17,19,40].
CD63 is another classical platelet activation marker. We reported elevated basal state platelet CD63 expression in both  the preeclampsia and normotensive pregnant women compared with in nonpregnant group indicated as higher CD63 positive platelet percent. Our study reconciled the results of previous studies [11,17,18,34], indicated that platelets from women with pregnant differed from platelets from nonpregnant women by expressing higher basal CD63 levels and being more responsive to in vitro agonist stimulation. Nevertheless, in contrast with our study, Holthe and Staff found there was no significant difference of basal CD63 expression between women with preeclampsia and NP women [10]. Differences in the study design possibly can explain this discrepancy.
During the study of the in vitro stimulated platelet response to TRAP-6, additional clues came forward that extended the information, which was yielded by the investigation of the basal platelet activation status alone. In substance, this approach contributed to discrimination between the pregnancy (women with preeclampsia/NP women) and the nonpregnant state, but in the case of TLT-1 also further distinguished the preeclampsia patients from normotensive pregnant women.
Regarding the soluble biomarkers, we found plasma concentration of sTLT-1 in PE group was markedly higher than in normotensive pregnant and nonpregnant women. And at a cutoff value of 46.1 pg/ml, sTLT-1 had sensitivity of 83.3% and specificity of 66.7% for preeclampsia diagnosis. The platelet activity implied as soluble P-selectin concentration between PE patients and NP women showed no significant difference. At a cutoff value of 85.0 ng/ml, soluble P-selectin was found to have sensitivity of 56.7% and specificity of 76.7% for preeclampsia development. These results are in line with the foundings of Marı´a E. Chavarrı´a et al., they reported that the cutoff value of soluble P-selectin for preeclampsia development was 81.5 ng/ml [41]. Moreover, the combination of sTLT-1 and sP-selectin markers may provide a more useful screening test than sP-selectin alone. The elevated TLT-1 expression levels both on platelet surface and soluble form in plasma further confirmed that higher TLT-1 levels may be a valuable indicator for platelet activation in preeclampsia.
One limitation of our study is based on a small sample of participants. We found the area under curve (AUC) of sTLT-1 in plasma was greater than AUC of sP-selectin, while without significant difference. More cases need to be recruited to verify whether soluble TLT-1 in plasma is preferable to sP-selectin as an indicator for platelet activation in preeclampsia. Furthermore, there are two sub-types including early and late onset preeclampsia, with others almost certainly yet to be identified. It is believed that pathogenetic mechanisms contributes in varying degrees in early and late onset pre-eclampsia. As reported, early and late onset preeclampsia are characterized by different biomarkers [28,32,33]. We didn't find the difference of concentration of sTLT-1 or sP-selectin between early and late onset preeclampsia patients. Our data were not able to further discriminate 12 cases of early and 18 cases of late onset preeclampsia in this study (unpublished data) for the finite cohort of 30 preeclamptic women. Further research should be undertaken to investigate the potential biomarkers of stratifying by preeclampsia type in future.
In conclusion, our data demonstrated that elevated TLT-1 expression levels both on platelet surface and soluble form in plasma are evident in preeclampsia when compared to normotensive pregnancy. Our results revealed that TLT-1 reflects platelet activation and may be a useful indicator for platelet activation in preeclampsia. These abnormalities of platelets may have implications for the pathogenesis of preeclampsia. Nevertheless, the exact pathogenic significance and clinical validity of TLT-1 in the maternal circulation needs to be better illustrated.

Disclosure statement
No potential conflict of interest was reported by the authors.

Author contributions
SJ, LH, and YZ designed the research, YZ, CH, ZZ collected and analyzed data. LH and YZ wrote the paper. LH, YZ, SJ performed experiments. QH helped to analyzed data. SJ and Q Huang initiated and supervised the project, analyzed and interpreted results. All authors reviewed the manuscript.