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Table of Contents
ORIGINAL ARTICLES
Year : 2021  |  Volume : 26  |  Issue : 2  |  Page : 118-122

Assessment of protein C antigen, free protein S, and protein C activity in pregnancy: A cross-sectional study of pregnant Nigerian women


1 Department of Haematology & Immunology, College of Medicine, University of Nigeria, Ituku Ozalla, Enugu, Nigeria
2 Department of Radiology, Abia State University Teaching Hospital, Aba Abia State, Nigeria
3 Department of Obstetrics & Gynaecology, College of Medicine, University of Nigeria, Ituku Ozalla, Enugu, Nigeria

Date of Submission30-Apr-2020
Date of Decision17-May-2020
Date of Acceptance21-Sep-2020
Date of Web Publication29-Jan-2021

Correspondence Address:
Theresa Ukamaka Nwagha
Department of Haematology & Immunology, College of Medicine, University of Nigeria, Ituku Ozalla, Enugu.
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmh.IJMH_21_20

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  Abstract 

Introduction: With increasing evidence of thromboembolic events among pregnant Nigerian women and associated high maternal mortality rates, there is a need to document the plasma levels of some markers of thrombosis in this population to aid prompt management of thromboembolic events. Objectives: To determine the plasma levels of free protein S (fPS), protein C (PC) antigen (PCAg), and PC activity (PCAc) in normal pregnancy, and any correlations with maternal age, gestational age (GA), and blood group. Materials and Methods: A cross-sectional study of eligible pregnant women receiving antenatal care in a tertiary hospital in south-south Nigeria. The plasma concentrations of fPS, PCAg, and PCAc were measured using enzyme-linked immunosorbent assay and Protac methods. Statistical analysis was both descriptive and inferential and done using SPSS, version 21, for windows. A P-value of <0.05 was considered statistically significant. Results: Eighty pregnant women at a GA of 25–42 weeks (mean 35.4 ±5.2) were recruited with a mean age of 30.4 ± 5.1 years. The mean plasma levels and range of fPS, PCAg, and PCAc were 47.2 ± 10.3%, 77.5 ± 23.2% and 110.4 ± 27.6%, respectively. There were significant positive correlations between PCAg and GA (r = 0.229, P = 0.041), PCAc and GA (r = 0.223, P = 0.046), and fPS and maternal age (r = 0.254, P = 0.023). Conclusion: Plasma concentration of PCAg and PCAc increased as pregnancy advanced, although fPS was below the reference limit, it increased with advancing maternal age. This information should be considered while evaluating pregnant women.

Keywords: Markers, Nigeria, pregnancy, thrombosis


How to cite this article:
Okoye HC, Nwagha TU, Ugwu AO, Eweputanna LI, Ugwu EO. Assessment of protein C antigen, free protein S, and protein C activity in pregnancy: A cross-sectional study of pregnant Nigerian women. Int J Med Health Dev 2021;26:118-22

How to cite this URL:
Okoye HC, Nwagha TU, Ugwu AO, Eweputanna LI, Ugwu EO. Assessment of protein C antigen, free protein S, and protein C activity in pregnancy: A cross-sectional study of pregnant Nigerian women. Int J Med Health Dev [serial online] 2021 [cited 2022 Aug 10];26:118-22. Available from: https://www.ijmhdev.com/text.asp?2021/26/2/118/308248




  Introduction Top


Protein C (PC) is a natural anticoagulant that exerts its anticoagulant activity by inactivating activated forms of coagulation factor FV and FVIII (FVa and FVIIIa). Protein S (PS) on the other hand brings to bear its anticoagulant property indirectly by serving as a cofactor to PC in the inactivation of FVa and FVIIIa and directly by binding to and inhibiting FXa, FVa, and FVIIIa.[1],[2] PC is thought to equally have some fibrinolytic properties by inhibiting plasminogen activator inhibitor type 1 (PAI-1) leading to a loss of the physiologic inhibition of tissue plasminogen activator (t-PA) making for more rapid activation of plasminogen.[3]

Normal pregnancy is regarded as a transient acquired thrombophilic state, with consequent coagulopathy and thrombosis. Plasma levels of coagulation factors such as FI, FVII, FVIII, FIX, FXI, and von Willebrand factor are increased during pregnancy in a bid to curb the hemostasis challenge associated with delivery.[4] At the same time, changes in the natural anticoagulants are observed as decrease in their plasma levels or development of resistance.[5] There is also a decrease in fibrinolytic activity due to reducing activity of t-PA during pregnancy.[6] This is further worsened by stasis and associated hormonal changes that occur during pregnancy. Other factors thought to increase the thrombogenic risks in pregnancy include, but not limited to, maternal age, obesity, multiple pregnancies, and mode of delivery.[7]

These tilt the balance of hemostasis during pregnancy to an increase in propensity to thrombosis and thromboembolic events. Non-communicable diseases like venous thromboembolism cardiovascular diseases continue to play an important, growing, and contributory etiologic role in maternal mortality.[8] The trend of maternal mortality has evolved through the years with thromboembolic events emerging as a leading cause of maternal death in developed countries.[9] Its contribution to maternal mortality rates in some low- and middle-income countries (LMIC) is slowly being recognized, especially now sustainable development goal (SDG) interventions are leading to a somewhat reduction in the other leading causes of maternal mortality in some LMIC.[10]

The literature is inconsistent with the changes in PCAg and PCAc during pregnancy.[11],[12],[13] In addition, there is a paucity of data on the changes in PCAg, PCAc, and fPS in pregnancy together with their relationship with maternal variables in our population. This study aims to determine the plasma concentrations of PCAg, PCAc, and fPS in normal Nigerian pregnant women, and to assess whether there are any correlations between these proteins and maternal age, gestational age (GA), and blood group.


  Materials and Methods Top


Study design and population

This is a cross-sectional study conducted in the University of Port Harcourt Teaching Hospital (UPTH) over a period of 3 months (February to April 2015). Eighty eligible pregnant women receiving antenatal care in the obstetrics unit of the hospital were recruited using a systematic sampling method. These women were further sub-grouped according to their GA. A sampling frame was made from a register of pregnant women who attended antenatal clinic (ANC) within the study period. Every first out of three registered names who presented for antenatal care and consented to participate were recruited for the study. Ethical approval was obtained from the Research and Ethics Committee of UPTH with ethics clearance certificate UPTH/ADM/90/S.IIVOL.X/234, and research was conducted according to the Helsinki declaration. Participants’ confidentiality was duly assured.

Information on their socio-demographics was sought for as well as details of their medical and obstetric history which was obtained from ANC case notes. The purpose of the study was explained after which only apparently healthy pregnant women who consented were included while those who had liver disease or any other chronic illness; diabetes, hypertension, asthma, cardiovascular disease; those being managed for venous thromboembolism; and those on anticoagulants were equally excluded from the study.

Sample collection and analysis

From each of the participants, 4.5mL of blood was collected and dispensed into sodium citrate bottle 0.5mL of 3.2% (nine parts of blood to one part of anticoagulant). Samples showing signs of hemolysis or with obvious clot formation were excluded from the analysis. Samples were centrifuged at 2500 RPM for 20 min after which the platelet-poor plasma was stored in aliquots at −20°C (freezers were connected to an alternative power source and daily temperature monitoring was done) until enough batch was pulled for determination of anticoagulant levels. The plasma concentrations of free PS (fPS), PC antigen (PCAg), and PC activity (PCAc) were measured by enzyme-linked immunosorbent assay (ELISA) and chromogenic methods using assay kits from Hyphen BioMed France (lot 130417A) and Technoclone Austria (lot 12021122; OK31B00.010), respectively. Plasma levels of these proteins were determined, and quality control measures were undertaken according to the respective manufacturer’s protocols with levels below 70% for fPS and PC correspondingly.

Statistics

Both descriptive and inferential analysis was done using the Statistical Package for Social Sciences (SPSS), version 21, for windows. Results were in tables and charts as means (standard deviation) and proportions where applicable. Pearson’s correlation coefficient was used to correlate plasma protein levels and age, parity, GA, gravity, and blood group. A P-value of <0.05 was considered statistically significant.


  Results Top


Socio-demographics

A total of 85 subjects were recruited for the study but 80 participants were analyzed (due to sample spillages). The mean age and GA of 30.4 ± 5.1 years and 35.4 ± 5.2 weeks, respectively. The median blood pressure was 110/70 mmHg. Subjects were further divided into groups with respect to age and GA. A fewest number of participants were below the age of 25 years while a majority (76.0%) were between 25 and 35 years of age. The highest number of subjects was between GA of 31 and 40 weeks. Details are shown in [Table 1] and [Table 2].
Table 1: Some demographics of study subjects

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Table 2: Descriptive statistics of some variables

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Association between the markers and some variables

Forty-eight (60.0%) of the study participants were of O-blood group and the majority (95.0%) were of Rh D positive blood group. The mean PCAg level and PC activity were 77.5 ± 23.2 and 110.4 ± 27.6, respectively, and within the reference range (70–150%) while the fPS mean level was 47.2 ± 10.3 and below the reference limit (70–150%) with a range between 27.1 and 63.1%.

Mean levels of the proteins did not differ significantly across age and blood groups [see [Table 3]]. On the other hand, FPS levels showed significantly higher levels in the older age group, r = 0.254, P = 0.023, while PCAg and PCAc significantly increased with increasing GA [see [Table 3]]. However, there is no significant association between gravidity, parity, blood group and plasma levels of these proteins [see [Table 4] and [Table 5]].
Table 3: Cross-tabulation of PCAg, PC activity, and fPS versus age

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Table 4: Cross-tabulation of PCAg, PC activity, and fPS versus blood group

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Table 5: Correlation of PC Ag, PC activity, and fPS with some variables

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FPS levels were lower than the reference range but remained consistently low across the GA [see [Figure 1]], whereas PCAg and PCAc levels slightly increased initially with increasing GA in the second trimester remained constant between 32 and 35 weeks GA then peaks again at 38 weeks after which it drops thereafter.
Figure 1: Relationship of markers of thrombosis and GA

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  Discussion Top


In a study of 80 pregnant women, we found that markers of thrombosis levels changed during pregnancy and were affected by some factors. Our study showed that fPS levels were consistently low during pregnancy; this confirmed results from other studies.[11],[12],[13] Our study also showed the fPS levels, although below the reference range, were higher before 28 weeks (48.2 ± 9.2%) and fell after 28 weeks only to peak at 37–40 weeks (49.4 ± 11.6%). Malm et al.[11] in a Swedish study showed similar trends although, in their study, they reported an overall reduction in the levels of PS antigen during pregnancy with the levels of fPS reduced to the lowest just before delivery, much less than what our study reported. An Indian study equally reported a similar free-fall of the fPS level comparable to levels seen in our study[13] Fraught et al.[12] in a Canadian study also reported a similar fall of fPS levels through the first and second trimesters with none recorded for the third trimester.

Although this fall in the fPS level is an adaptive physiologic change in pregnancy, its mechanism is largely unknown. The values of fPS levels and the degree of fall seen in our study when compared to reports from other studies[11],[12] could suggest genetic or racial disparity. Further studies are needed to clarify this issue.

We also observed a significant association between fPS levels and maternal age; the significance of this is unknown as there is no association between the fPS and maternal age in the reviewed literature. Again further studies can help answer this.

The present study shows that PCAg and PCAc levels increased during pregnancy. Our findings agree with reports from other studies.[12],[14] PCAg and PCAc levels significantly increased with increasing GA. The trend in our study showed levels remained constant between 32 and 35 weeks peaked at 38 weeks after which it dropped. Although a Canadian study by Fraught et al.[12] reported an increase in the levels of PCA throughout pregnancy, this increase was not significantly associated with GA. Warwick et al. in a UK study reported somewhat similar findings as their study showed a significant rise in PCA only in the second trimester.[15] Nwagha et al. in a study done in Enugu, Nigeria, equally reported a similar significant trend in PCAc levels with a slight drop in the third trimester with an increase in acquired PC resistance during pregnancy.[16] We did not measure PC resistance in this study.

Brenner in his review posited that PCAc levels are a well-established adaptive change seen in pregnancy although PCAg levels remain unchanged.[17] Our study and similar studies[11],[12],[13] have reported an increase in PCAg and PCAc levels during pregnancy. Changes in these protein levels, increase in procoagulant factors like VIII, IX, X and diminishing fibrinolytic protein levels attributed to high estrogen levels makes pregnancy a hypercoagulable in preparation of intrapartum haemostatic challenge.

The study observed only one participant with a very low level of fPS (27.1%), who would benefit from thrombophilic screening after her pregnancy. The changes observed in these markers of thrombosis may add on to an increased risk of pregnancy-associated venous thromboembolic events in our pregnant women with its possible contribution to an increase in maternal mortality and morbidity in our environment.

Limitations

There was no correlation of the levels of these markers with a clinical presentation during pregnancy or pregnancy outcome, PC resistance was not measured in these women, and these markers (PCAg, PCAc, and fPS) were not measured in the puerperium. There was no pre-pregnancy measurement of these markers of thrombosis to better appreciate these changes in pregnancy and the possible prevalence of inherited or acquired thrombophilia among these women.


  Conclusion Top


Markers of thrombosis (PCAg, PCAc, and fPS) change during pregnancy, PCAg, and PCAc levels increased significantly throughout gestation, and there was a nonsignificant low level of fPS during pregnancy. This is may contribute to hypercoagulability of pregnancy. Hence, a high index of clinical suspicion for pregnancy-associated VTE events among our pregnant women and is key. The obtained plasma levels may be a useful guide for the clinicians when ordering levels of these markers during pregnancy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Mosnier LO, Griffin JH Protein C anticoagulant activity in relation to anti-inflammatory and anti-apoptotic activities. Front Biosci 2006;11:2381-99.  Back to cited text no. 1
    
2.
Okoye HC, Eweputanna LI, Okpani AO, Ejele OA Associations between pre-eclampsia and protein C and protein S levels among pregnant Nigerian women. Int J Gynaecol Obstet 2017;137:26-30.  Back to cited text no. 2
    
3.
Mosnier LO, Zlokovic BV, Griffin JH The cytoprotective protein C pathway. Blood 2007;109:3161-72.  Back to cited text no. 3
    
4.
Prisco D, Ciuti G, Falciani M Haemostatic changes in normal pregnancy. Haematol Rep 2005;1:1-5.  Back to cited text no. 4
    
5.
Cumming AM, Tait RC, Fildes S, Yoong A, Keeney S, Hay CR Development of resistance to activated protein C during pregnancy. Br J Haematol 1995;90:725-7.  Back to cited text no. 5
    
6.
Ishii A, Yamada S, Yamada R, Hamada H T-PA activity in peripheral blood obtained from pregnant women. J Perinat Med 1994;22:113-7.  Back to cited text no. 6
    
7.
Virkus RA, Løkkegaard E, Lidegaard Ø, Langhoff-Roos J, Nielsen AK, Rothman KJ, et al. Risk factors for venous thromboembolism in 1.3 million pregnancies: A nationwide prospective cohort. PLoS One 2014;9:e96495.  Back to cited text no. 7
    
8.
Maternal Health Task Force. The sustainable development goal and maternal mortality. https://www.mhtf.org/topics/the-sustainable-development-goals-and-maternal-mortality/. Accessed 29 April 2020.  Back to cited text no. 8
    
9.
Creanga AA, Berg CJ, Syverson C, Seed K, Bruce FC, Callaghan WM Pregnancy-related mortality in the United States, 2006–2010. Obstet Gynecol 2015;125:5-12. doi:10.1097/AOG.0000000000000564  Back to cited text no. 9
    
10.
Tessema GA, Laurence CO, Melaku YA, Misganaw A, Woldie SA, Hiruye A, et al. Trends and causes of maternal mortality in Ethiopia during 1990–2013: Findings from the global burden of diseases study 2013. BMC Public Health 2017;17:160.  Back to cited text no. 10
    
11.
Malm J, Laurell M, Dahlbäck B Changes in the plasma levels of vitamin K-dependent proteins C and S and of c4b-binding protein during pregnancy and oral contraception. Br J Haematol 1988;68:437-43.  Back to cited text no. 11
    
12.
Faught W, Garner P, Jones G, Ivey B Changes in protein C and protein S levels in normal pregnancy. General Obstet Gynecol 1995;172:147-50.  Back to cited text no. 12
    
13.
Deren BA, Buyukasik YO, Basaran M Free protein s reference ranges in gravidas without hereditary and acquired thrombophilia. Indian J Hematol Blood Transfus 2015;31:286-91. doi:10.1007/s12288-014-0448-3  Back to cited text no. 13
    
14.
Warwick R, Hutton RA, Goff L, Letsky E, Heard M Changes in protein C and free protein S during pregnancy and following hysterectomy. J R Soc Med 1989;82:591-4.  Back to cited text no. 14
    
15.
Nwagha UT, Nwagha UI, Ibegbulam OG, Ocheni S, Okpala I, Ezeonu PO, et al. Increased prevalence of activated protein C resistance during pregnancy may implicate venous thrombo embolic disorders as a common cause of maternal mortality in Nigeria. J Basic Clin Reprod Sci 2012;1:19-24.  Back to cited text no. 15
  [Full text]  
16.
Brenner B Haemostatic changes in pregnancy. Thromb Res 2004;114:409-14.  Back to cited text no. 16
    
17.
Franchini M Haemostasis and pregnancy. Thromb Haemost 2006;95:401-13.  Back to cited text no. 17
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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