Decreasing Platelet Aggregation Despite Increasing Soluble P-selectin during Pregnancy in Women with and without Heterozygous Factor V Leiden Mutation

Abstract

Background

Risk of venous thromboembolism (VTE) is increased in pregnancy and postpartum, and 40% of VTEs in pregnancy (Caucasians) are associated with heterozygous factor V Leiden mutation (FVL). Thrombin generation is increased in individuals with FVL and in pregnant women, and thrombin amplifies both platelet and coagulation activation. Although both contribute to VTE pathophysiology, the mechanisms of platelet activation in pregnant women, particularly with heterozygous FVL, remain poorly understood.

Objectives

To describe the physiological course of the platelet activation marker plasma soluble P-selectin (sP-selectin), whole blood platelet aggregation, and thromboelastography (TEG) parameters throughout pregnancy and postpartum, and assess differences between women with and without heterozygous FVL.

Patients/Methods

A total of 22 pregnant women with heterozygous FVL and 22 without were enrolled. Blood samples were collected at multiple time points during and after pregnancy. Platelet activation and aggregation were evaluated using sP-selectin, multiple electrode aggregometry (MEA) with adenosine diphosphate, arachidonic acid, thrombin receptor-activating peptide-6 as agonists, and TEG.

Results

sP-selectin levels increased significantly during pregnancy, while platelet aggregation decreased in response to all agonists (P < 0.005). TEG maximum amplitude (MA) increased throughout pregnancy. No significant differences were observed between women with and without FVL.

Conclusion

In late pregnancy, decreased platelet aggregation responses were observed alongside increased sP-selectin levels, with no differences in levels between women with and without heterozygous FVL. These findings indicate that the presence of heterozygous FVL does not significantly influence platelet function during pregnancy. The cause of the unexpected, reduced platelet aggregation remains unclear and warrants further investigation.

Keywords

platelet aggregation - pregnancy - P-selectin - thrombophilia - venous thromboembolism

Introduction

Pregnancy and the postpartum period are well-established prothrombotic states with a 6-fold and 22-fold increased risk of venous thromboembolism (VTE), respectively.[1] VTE occurs in approximately 1.2 per 1,000 deliveries[2] and remains a leading cause of maternal mortality, contributing to 17.0% of maternal deaths in developed countries and 7.4% globally.[3] This elevated risk is driven by physiological changes including increased levels of coagulation factors, reduced anticoagulant activity, impaired fibrinolysis, venous stasis, and endothelial injury.[4] The presence of inherited thrombophilia further amplifies this risk. Heterozygous factor V Leiden (FVL) mutation is the most frequent inherited thrombophilia, with a prevalence ranging from 3 to 15% in Caucasians, and the highest rates are observed in Northern and Eastern Europe.[5] The mutation prevents efficient inactivation of factor Va by activated protein C (APC),[5] in addition to impaired FVa inactivation by tissue factor pathway inhibitor,[6] leading to sustained thrombin generation and increased VTE risk. In Caucasians, FVL can be found in approximately 40% of the pregnancy-associated VTEs.[7]

Thrombin is known to potentiate platelet activation,[8] and platelets play a central role in VTE pathogenesis.[9] Activated platelets initiate thrombus formation by adhering to injured endothelium and recruiting leukocytes, which release neutrophil extracellular traps (NETs), forming a scaffold for thrombus development. Activated platelets also expose phosphatidylserine, providing a surface for thrombin generation, and release procoagulant microparticles and active substances from their granula. Increased thrombin generation is found both in healthy pregnancies[10] [11] and in non-pregnant individuals with FVL.[12] [13] However, whether thrombin generation and platelet activation are further elevated in pregnant women with FVL compared with those without remains uncertain. Previous studies have been inconclusive and confounded by including women using low-molecular-weight heparin (LMWH).[14] [15]

Platelet aggregation assays such as multiple electrode aggregometry (MEA) and viscoelastic methods like thromboelastography (TEG) offer insights into platelet function and clot strength, but data in pregnancy are limited and conflicting.[16] Interpretation in pregnancy may be difficult because physiological changes in pregnancy (e.g., hemodilution, reduced platelet count, and increased fibrinogen) may affect measurements of platelet aggregation differently, depending on methods.[17] [18] [19] [20] Soluble P-selectin (sP-selectin), shed from activated platelets, is an established marker of in vivo platelet activation[21] [22] and is suggested to induce NET formation,[23] [24] a key mechanism implicated in the pathogenesis of VTE.[25] Elevated sP-selectin levels have been associated with VTE in men and non-pregnant women.[26] [27] Elevated levels have also been found in pregnancy, both healthy[28] and complicated by preeclampsia,[29] but whether it is higher in pregnant women with thrombophilia is not known.

Despite the known prothrombotic state of pregnancy and the established role of FVL in increasing VTE risk, there is a lack of comprehensive data on platelet activation and aggregation in pregnant women with thrombophilia. We hypothesize that platelet activation is increased during pregnancy and postpartum, and that this activation is further amplified in women with heterozygous FVL due to enhanced thrombin generation and platelet responsiveness. Our study objectives are therefore to characterize the course of platelet activation during pregnancy and postpartum using sP-selectin and MEA, and to evaluate clot strength and thrombin-related platelet activity using TEG, and, in addition, to compare these parameters in women with and without heterozygous FVL.

Methods

Study Participants

The Graviclot study is a prospective cohort study, conducted in 2017 to 2020. Pregnant women (controls) and pregnant women with heterozygous FVL (c.1601G > A) and/or other risk factors for VTE, including presence of the heterozygous prothrombin gene point mutation (c*97G > A) (PGM), protein C or S deficiency, and/or family history of VTE (at least one first-degree relative with VTE before the age of 50 years), were included before the 14th week of gestation. Exclusion criteria were twin pregnancy and planned anticoagulant prophylaxis during pregnancy.

Study participants were recruited via their primary care physician or through an information campaign in social media and on the Haukeland University Hospital webpage. Health information regarding the presence of thrombophilia, family history of thrombosis, blood type, bleeding tendency, chronic disease, and medication was collected at the first time point for blood draw, and information regarding conditions that could possibly influence the test results (e.g., recent acute illness, changes in medication, pregnancy complications, and increased bleeding tendency) was registered at each blood drawing. Altogether, 22 women without inherited risk factors for VTE (controls), 22 with heterozygous FVL, and 12 with other thrombophilia and/or family history of VTE were included in their first trimester of pregnancy. All participants were Caucasians.

The study was approved by the regional ethics committee (REK-ID 2016/1083) and written informed consent was obtained from each participant.

Blood Sampling

Blood samples were obtained at four different time points during pregnancy (gestational weeks 12–14, 20–22, 28–30, and 36–38), and three times postpartum (at delivery [0–1 days postpartum], 2 weeks, and 8–10 weeks postpartum). Venous blood sampling was performed after 15 minutes of rest, with a butterfly device. Blood samples were collected in lithium heparin tubes for MEA analysis (use of tubes validated by Peerschke et al[30] and reference intervals given by Roche), sodium citrate 3.2% (0.109 M) tubes for fibrinogen, sP-selectin, and TEG, and EDTA-K2 tubes (all Vacuette, Greiner Bio-One, Kremsmünster, Austria) for hematology analyses. Double centrifugation, 2,500 × g for 15 minutes at room temperature, were performed to gain platelet-free citrated plasma that was aliquoted and frozen at −80°C within 2 hours after blood sampling.

Analysis

The hematological parameters hemoglobin, hematocrit, platelet count, and mean platelet volume (MPV) were analyzed by Advia 2120i (Siemens Healthineers, Erlangen, Germany). Fibrinogen (STA Fibrinogen) and antithrombin (AT) (STA Antithrombin) were analyzed by STA-R Max (Stago, Asnières-sur-Seine, France), and Protein S free (PS) (Innovance Protein S free) and Protein C (PC) (Berichrom Protein C) were analyzed by Sysmex CS-5100 (Siemens Healthineers, Marburg, Germany). Chromogenic anti-FXa activity was performed with STA-Liquid Anti-Xa reagents (Stago) on ACL TOP 700 (Werfen, MA, USA). Point mutation gene analyses of FVL (c.1601G > A) and PGM (c*97G > A) were performed by loop-mediated isothermal amplification (LAMP) on Light Cycler 480 II (Roche Diagnostics GmbH, Mannheim, Germany).

Concentration of sP-selectin was measured with the Quantikine ELISA Human P-selectin/CD62P Immunoassay (R&D Systems, MN, USA) according to the manufacturer's instructions, but using twice frozen/thawed citrated plasma, which may moderately decrease sP-selectin levels.[31] Results are presented not only as concentrations (ng/mL), but also as the ratio between sP-selectin concentration and the corresponding platelet count at each sampling.

Platelet aggregation was measured with MEA (Multiplate analyzer, Roche Diagnostics GmbH). All samples were analyzed between 30 and 60 minutes after sampling. Samples containing 300 µL 0.9% saline and 300 µL heparinized whole blood were constantly stirred and incubated at 37°C for 3 minutes. The diluted whole blood samples were activated with 20 µL of agonist, either adenosine diphosphate (ADP), arachidonic acid (ASPI), or thrombin receptor-activating peptide-6 (TRAP) with final concentrations of 6.5, 0.5, and 32 µM, respectively, according to manufacturer's instructions. Aggregation was recorded for 6 minutes and quantified and presented as the area under the aggregation-curve (AUC) in arbitrary units (U). The aggregation results were also adjusted for the corresponding hematocrit at each sampling and normalized (e.g., ADP/hematocrit × 0.4).

The viscoelastic hemostatic properties, time to first clot formation (R), clot formation time (K), rate of clot formation (α-angle), maximum clot strength, represented by maximum amplitude (MA), coagulation index (CI), and clot lysis at 30 minutes after maximum clot strength (LY30), were measured between 30 and 60 minutes after blood drawing using the TEG 5000 thromboelastograph (Haemonetics Corporation, Braintree, MA, USA). After activating 1 mL of citrated whole blood with 40 µL of kaolin, 340 µL was transferred to a cup containing 20 µL of 0.2 M CaCl₂. Cups containing heparinase were used at time points with LMWH administration. The analysis ran until 30 minutes after reaching MA.

Statistics

The pregnant women were divided into three groups characterized by the absence or presence of risk factors for VTE: (1) women without inherited risk factors or family history of VTE (controls), (2) women with heterozygosity for FVL with or without family history (FVL group), and (3) women with other thrombophilia and/or family history (others) (see Study Participants in Methods and [Table 1]). Evaluation of normal distribution was performed by visual assessment (Q-Q plots) and the Shapiro-Wilk test, and medians (2.5–97.5 percentiles) and means (−/+ 2 standard deviations [SD]) were calculated for each time point. All data were normally distributed, except TEG LY30. Comparisons were performed using mixed models, applying Dunnett's correction for multiple comparisons for continuous variables over time and Sidák's correction for group comparison at each time point, except for TEG LY30 where Mann-Whitney U test was used. Group comparisons were performed only between controls and the FVL group, as the others group was small and heterogenous. Pearson correlation coefficient was used for correlation analysis. Differences in age and body mass index (BMI) between groups were tested with the unpaired t-test. Outliers were detected by Grubb's test. A P value of < 0.05 was considered statistically significant in all analyses. GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, USA) was used for data management and statistical analysis.

Table 1 Characteristics of study participants

	Controls (n = 22)	Heterozygous factor V Leiden mutation (n = 22)	Other thrombophilia (n = 12)
Maternal characteristics/preexisting risk factors
Age (years) at inclusion
Median (min; max)	32 (21; 38)	29 (20; 36)	32 (23; 39)
Age > 35 years (percentage)	7 (31.8%)	2 (9.1%)	1 (8.3%)
BMI (kg/m2) at time of inclusion
Median (min; max)	24 (19; 32)	26 (20; 36)	24 (21; 36)
Obesity (BMI ≥ 30 kg/m2)	4 (18.2%)	5 (22.7%)	1 (8.3%)
Parity
0	8 (36.4%)	11 (50.0%)	4 (33.3%)
1	8 (36.4%)	7 (31.8%)	7 (58.3%)
2	6 (27.3%)	4 (18.2%)	1 (8.3%)
ABO blood group
A	11 (50.0%)	11 (50.0%)	5 (41.7%)
AB	1 (4.5%)	0 (0%)	0 (0%)
Family history of VTE (in first-degree relative)	0 (0%)	8 (36.4%)	8 (66.7%)
Inherited thrombophilias
Factor V Leiden mutation (c.1601G > A), heterozygous	–	22 (100%)	–
Prothrombin mutation (c*97G > A), heterozygous	–	–	3 (25.0%)
Protein S deficiency	–	–	3 (25.0%)
Protein C deficiency	–	–	1 (8.3%)
Family history of VTE (in first-degree relative) only[a]	–	–	5 (41.7%)
Pregnancy characteristics/obstetric risk factors
Gestational diabetes	2 (9.1%)	2 (9.1%)	0 (0%)
Preeclampsia	3 (13.6%)	0 (0%)	0 (0%)
Assisted reproductive technique	1 (4.5%)	0 (0%)	2 (16.7%)
Hyperemesis	0 (0%)	2 (9.1%)	0 (0%)
Acetylsalicylic acid (only in pregnancy)	0 (0%)	3 (13.6%)	0 (0%)
Delivery/postpartum characteristics
Transfusion	0 (0%)	0 (0%)	1 (8.3%)
Peripartum hemorrhage (>1 L)	2 (9.1%)	1 (4.5%)	3 (25.0%)
Postpartum infection	8 (36.4%)	8 (36.4%)	4 (33.3%)
Preterm delivery, <37 weeks	0 (0%)	2 (9.1%)	0 (0%)
Caesarean section	3 (13.6%)	1 (4.5%)	2 (16.7%)
Low-molecular-weight heparin (only postpartum)	4 (18.2%)	8 (36.4%)	4 (33.3%)

Abbreviations: BMI, body mass index; VTE, venous thromboembolism.

Note: For the continuous variables age and BMI, the median (min; max) is presented. For the categorical variables, numbers (percentages) are presented.

^a No other known thrombophilia.

Results

Participants

Baseline characteristics of the 56 women and information about their health status during pregnancy are presented in [Table 1]. Age (mean 31 years, min-max 20–39) and BMI (25 kg/m² [19–36]) at inclusion did not differ between controls and the FVL group (p = 0.158 and 0.283, respectively); however, seven controls and only two in the FVL group were > 35 years. Eight (36.4%) women in the FVL group had family history of VTE in at least one first-degree relative. Preeclampsia in mild form was found at delivery in three controls and none in the FVL group. Overall, the number of other risk factors for VTE related to pregnancy and delivery was approximately equally distributed in controls and the FVL group.

Three women in the FVL group used prophylactic acetylsalicylic acid (ASA, Albyl-E, Orifarm Healthcare) 75 mg d⁻¹ throughout pregnancy (none postpartum). Postpartum, prophylactic treatment with LMWH was used by 16 of the women (4 controls mainly due to caesarean section, 8 in the FVL group, and 4 in others), with treatment periods ranging from 1 day to 7 weeks after delivery. However, anti-Xa results, indicating LMWH presence in the blood, were only found in three samples at delivery and three samples 2 weeks postpartum (range 0.10–0.39 IU/mL).

Missing Data

As two participants withdrew from the study, a total of 56 were included ([Supplementary Fig. S1], available in the online version only). While 1 missed three planned time points, 11 participants lacked results for one of the time points. Reasons for this were late inclusion (after gestational week 14), cancellation due to COVID-19 restrictions, pre-term birth, or logistical issues for participants or the laboratory, especially for deliveries during weekends or holidays. A few single results for hematology, MEA, and TEG are missing due to registration errors or logistical issues (weekends/holidays).

Laboratory Results

Hematology Analyses and Fibrinogen during Pregnancy and Postpartum

Hemoglobin and hematocrit decreased as expected during pregnancy (physiological hemodilution) from mean 12.9 to 12.1 g/dL and 0.37 to 0.36 L/L, respectively, with the lowest levels at delivery (mean 11.5 g/L and 0.34 L/L), and normalization 2 weeks postpartum ([Table 2]). Platelet counts decreased slightly during pregnancy from 242 × 10⁹/L to 231 × 10⁹/L, with lower levels at delivery (217 × 10⁹/L). Platelet counts exceeded 100 × 10⁹/L for all participants at all time points. The platelets reached peak level at 2 weeks postpartum with 337 × 10⁹/L. MPV and fibrinogen increased throughout pregnancy, from 8.3 to 9.7 fL and 4.0 to 4.9 g/L, respectively, reaching maximum during the last trimester and at delivery, with most results within reference intervals at 8 to 10 weeks postpartum ([Table 2]). One sample with platelet count of 1,116 × 10⁹/L 2 weeks postpartum (systemic infection) was considered an outlier, and this result was therefore excluded.

Table 2 Hemoglobin, hematocrit, platelet count, mean platelet volume (MPV), fibrinogen, soluble P-selectin (sP-selectin), sP-selectin divided by platelet count, and platelet aggregation determined by multiple electrode impedance aggregometry after activation by adenosine diphosphate (ADP), arachidonic acid (ASPI), and trap-6 (TRAP) in pregnancy and postpartum (pp)

Analyte RI, non-pregnant	Pregnancy				Postpartum
	12–14 weeks	20–22 weeks	28–30 weeks	36–38 weeks	Delivery	2 weeks pp	8–10 weeks pp
Hemoglobin 11.7–15.3 g/dL[a]	Med (2.5–97.5) Mean (−/+ 2 SD)	Med (2.5–97.5) Mean (−/+ 2 SD)	Med (2.5–97.5) Mean (−/+ 2 SD)	Med (2.5–97.5) Mean (−/+ 2 SD)	Med (2.5–97.5) Mean (−/+ 2 SD)	Med (2.5–97.5) Mean (−/+ 2 SD)	Med (2.5–97.5) Mean (−/+ 2 SD)
All, n = 56	12.7 (11.3–14.6) 12.9 (11.3–14.5)	12.2 (10.8–13.9) 12.3 (10.9–13.7)*	11.9 (10.2–13.7) 11.9 (10.3–13.5)*	12.1 (9.9–14.6) 12.1 (9.9–14.3)*	11.3 (9.2–13.9) 11.5 (9.3–13.7)*	13.0 (10.5–15.5) 12.9 (10.5–15.3)	13.0 (10.9–14.5) 13.0 (11.2–14.8)
	Med (min; max)	Med (min; max)	Med (min; max)	Med (min; max)	Med (min; max)	Med (min; max)	Med (min; max)
Controls, n = 22	13.0 (12.0; 14.2)	12.3 (11.4; 13.6)	11.9 (10.1; 13.9)	12.2 (9.8; 14.7)	11.6 (9.3; 13.7)	12.8 (10.6; 14.6)	12.9 (11.6; 14.4)
FVL, n = 22	12.6 (11.5; 14.8)	12.3 (11.0; 13.9)	12.2 (10.4; 13.4)	12.3 (10.0; 14.5)	11.8 (9.2; 14.0)	13.4 (10.4; 15.7)	13.1 (10.8; 14.6)
Others, n = 12	12.6 (11.1; 14.0)	11.8 (10.7; 13.3)	11.8 (10.6; 12.8)	11.9 (10.7; 13.4)	11.1 (9.9; 12.0)	13.3 (10.8; 14.9)	13.2 (11.4; 13.8)
Hematocrit 0.35–0.46 L/L[a]
All, n = 56	0.37 (0.32–0.43) 0.37 (0.32–0.42)*	0.36 (0.31–0.40) 0.36 (0.32–0.40)*	0.35 (0.29–0.41) 0.35 (0.30–0.40)*	0.35 (0.29–0.42) 0.36 (0.30–0.42)*	0.34 (0.27–0.42) 0.34 (0.28–0.40)*	0.39 (0.32–0.49) 0.39 (0.32–0.46)	0.40 (0.34–0.44) 0.39 (0.34–0.44)
Controls, n = 22	0.37 (0.33; 0.41)	0.36 (0.33; 0.39)	0.34 (0.29; 0.39)	0.35 (0.29; 0.41)	0.34 (0.27; 0.39)	0.39 (0.32; 0.43)	0.40 (0.35; 0.43)
FVL, n = 22	0.37 (0.32; 0.43)	0.36 (0.32; 0.40)	0.35 (0.30; 0.42)	0.36 (0.30; 0.42)	0.35 (0.28; 0.43)	0.40 (0.32; 0.51)	0.40 (0.34; 0.44)
Others, n = 12	0.37 (0.32; 0.41)	0.35 (0.31; 0.38)	0.35 (0.31; 0.38)	0.35 (0.33; 0.40)	0.33 (0.30; 0.37)	0.40 (0.35; 0.44)	0.39 (0.35; 0.42)
Platelet count 165–387 × 109/L[a]
All, n = 56	238 (138–370) 242 (142–342)*	238 (132–403) 246 (132–360)*	241 (128–375) 246 (134–358)*	228 (121–357) 231 (119–343)*	215 (120–382) 217 (107–327)*	330 (196–586) 337 (165–509)*	253 (164–378) 261 (155–367)
Controls, n = 22	242 (176; 307)	236 (180; 324)	240 (168; 350)	218 (152; 361)	222 (121; 400)	339 (215; 565)	252 (176; 348)
FVL, n = 22	239 (131; 346)	239 (127; 374)	236 (115; 366)	232 (108; 315)	208 (120; 294)	339 (190; 471)	252 (156; 383)
Others, n = 12	228 (159; 385)	237 (165; 424)	254 (147; 381)	237 (157; 292)	224 (143; 306)	306 (221; 597)	256 (189; 357)
MPV 7.0–9.0 fL[b]
All, n = 56	8.2 (6.5–10.8) 8.3 (6.2–10.4)	8.3 (6.7–11.2) 8.6 (7.4–10.8)	8.4 (7.1–11.9) 8.8 (6.5–11.1)	9.7 (7.4–13.9) 9.7 (6.7–12.7)*	9.7 (7.3–13.9) 9.9 (6.7–13.1)*	8.1 (6.5–11.1) 8.3 (6.0–10.6)	8.5 (6.6–11.3) 8.6 (6.2–11.0)
Controls, n = 22	7.6 (6.4; 8.6)	7.9 (6.4; 9.9)	8.1 (7.0; 10.5)	9.1 (7.3; 11.8)	8.9 (7.3; 12.7)	7.5 (6.4; 9.9)	7.8 (6.5; 9.3)
FVL, n = 22	8.7 (7.2; 11.1)†	9.2 (7.6; 11.2)†	8.8 (7.4; 12.2)	10.2 (7.7; 14.4)	10.3 (7.9; 14.0)	8.8 (6.7; 11.3)†	9.3 (7.0; 11.1)†
Others, n = 12	8.0 (6.6; 10.1)	8.5 (7.5; 10.9)	9.1 (7.2; 10.8)	9.9 (7.5; 11.5)	10.2 (8.5; 11.1)	8.7 (7.1; 9.8)	8.6 (6.9; 11.4)
Fibrinogen 2.0–4.0 g/L[a]
All, n = 56	3.9 (3.2–5.5) 4.0 (2.9–5.1)*	3.9 (3.1–5.5) 4.0 (2.9–5.1)*	4.2 (3.2–6.0) 4.3 (3.0–5.6)*	4.8 (3.5–6.2) 4.9 (3.6–6.2)*	4.9 (3.2–6.1) 4.8 (3.3–6.3)*	3.4 (2.3–5.9) 3.6 (2.1–5.1)*	3.0 (2.1–4.1) 3.0 (2.1–3.9)
Controls, n = 22	3.7 (3.2; 4.9)	3.6 (3.0; 4.9)	4.1 (3.2; 5.5)	4.7 (3.9; 6.2)	4.7 (3.4; 6.2)	3.5 (2.3; 6.3)	2.9 (2.0; 4.0)
FVL, n = 22	4.0 (3.2; 5.5)	4.0 (3.1; 5.6)	4.2 (3.3; 6.2)	4.8 (3.4; 6.3)	4.9 (3.2; 6.0)	3.4 (2.4; 5.3)	3.1 (2.3; 4.1)
Others, n = 12	4.0 (3.6; 5.4)	4.1 (3.4; 5.2)	4.5 (3.9; 5.5)	4.9 (3.9; 5.5)	5.0 (3.1; 5.6)	3.4 (3.1; 4.5)	2.8 (2.6; 3.8)
sP-selectin <38 ng/mL[b]
All, n = 56	20 (7–33) 20 (8–32)	25 (8–41) 24 (10–37)*	25 (8–49) 25 (8–41)*	26 (10–72) 28 (5–50)*	32 (11–69) 35 (10–60)*	26 (8–44) 27 (10–43)*	19 (6–32) 20 (6–32)
Controls, n = 22	17 (7; 34)	22 (6; 41)	22 (7; 50)	22 (9; 45)	30 (9; 66)	25 (8; 40)	17 (6; 33)
FVL, n = 22	21 (12; 32)	25 (17; 40)	25 (13; 40)	28 (21; 42)	39 (22; 57)	27 (17; 43)	20 (13; 32)
Others, n = 12	21 (13; 29)	26 (17; 33)	25 (19; 47)	28 (18; 85)	34 (18; 71)	26 (16; 44)	24 (14; 31)
sP-selectin/ platelet count
All, n = 56	0.08 (0.03–0.16) 0.09 (0.03–0.15)	0.10 (0.03–0.20) 0.10 (0.03–0.17)*	0.10 (0.03–0.23) 0.11 (0.03–0.19)*	0.12 (0.04–0.31) 0.13 (0.02–0.24)*	0.15 (0.05–0.36) 0.17 (0.02–0.32)*	0.08 (0.03–0.17) 0.08 (0.02–0.14)	0.08 (0.03–0.14) 0.08 (0.05–0.13)
Controls, n = 22	0.08 (0.03; 0.16)	0.10 (0.03; 0.14)	0.09 (0.03; 0.18)	0.12 (0.04; 0.17)	0.13 (0.04; 0.27)	0.08 (0.02; 0.13)	0.07 (0.02; 0.13)
FVL, n = 22	0.08 (0.04; 0.16)	0.10 (0.06; 0.21)	0.11 (0.04; 0.24)	0.12 (0.08; 0.24)	0.18 (0.10; 0.38)	0.09 (0.04; 0.18)	0.08 (0.04; 0.15)
Others, n = 12	0.08 (0.06; 0.14)	0.10 (0.05; 0.13)	0.12 (0.06; 0.14)	0.12 (0.09; 0.35)	0.14 (0.12; 0.28)	0.08 (0.06; 0.14)	0.09 (0.07; 0.11)
ADP 55–117 U[b]
All, n = 56	71 (45–130) 74 (37–112)	58 (26–106) 60 (24–96)*	45 (17–81) 46 (18–73)*	43 (19–73) 45 (17–72)*	63 (21–110) 65 (24–107)	73 (45–131) 78 (36–120)*	66 (43–111) 67 (39–96)
Controls, n = 22	77 (44; 97)	62 (28; 83)	44 (23; 74)	40 (21; 74)	66 (25; 102)	78 (56; 133)	61 (41; 114)
FVL, n = 22	71 (48; 122)	54 (25; 103)	48 (12; 86)	46 (18; 70)	64 (20; 111)	73 (51; 113)	67 (52; 91)
Others, n = 12	63 (48; 134)	56 (45; 108)	47 (24; 63)	47 (25; 57)	63 (30; 92)	71 (45; 112)	66 (47; 85)
ASPI 79–141 U[b]
All, n = 56	94 (35–152) 97 (46–148)*	74 (27–122) 76 (32–108)*	61 (23–105) 59 (24–95)*	52 (16–96) 56 (16–96)*	64 (16–124) 70 (46–125)*	96 (50–156) 96 (48–145)*	84 (49–125) 86 (50–122)
Controls, n = 22	96 (58; 136)	83 (41; 106)	64 (34; 115)	49 (25; 92)	71 (28; 125)	97 (51; 161)	85 (55; 116)
FVL, n = 22	96 (30; 157)	64 (25; 118)	62 (23; 91)	52 (14; 78)	64 (32; 116)	96 (49; 136)	84 (46; 128)
Others, n = 12	89 (66; 143)	75 (54; 124)	53 (30; 80)	56 (20; 97)	63 (13; 119)	91 (65; 142)	82 (59; 106)
TRAP 92–151 U[b]
All, n = 56	113 (76–162) 115 (70–160)	95 (42–138) 94 (49–139)*	71 (22–108) 71 (32–111)*	67 (28–116) 67 (24–110)*	85 (39–163) 90 (36–143)*	118 (75–170) 118 (70–165)	110 (72–146) 110 (75–145)
Controls, n = 22	113 (76; 157)	96 (59; 128)	70 (48; 104)	66 (36; 119)	88 (45; 125)	122 (86; 170)	113 (68; 149)
FVL, n = 22	120 (77; 163)	91 (39; 138)	76 (23; 108)	67 (25; 96)	85 (38; 169)	118 (74; 158)	110 (82; 134)
Others, n = 12	108 (82; 159)	99 (63; 138)	67 (21; 97)	65 (43; 101)	91 (43; 132)	106 (81; 159)	107 (91; 139)

Abbreviations: All, all study participants; Controls, participants without thrombophilia; FVL, participants with heterozygous factor V Leiden mutation; max, maximum; Med, median; min, minimum; Others, participants with other inherited thrombophilia (heterozygous prothrombin mutation, protein S or C deficiency) or family history of venous thromboembolism; RI, reference intervals; SD, standard deviation.

Note: Statistically significant differences (p < 0.05) from 8 to 10 weeks pp are marked with *, and between controls and FVL with †.

^a Haukeland University Hospital, non-pregnant women >18 years.

^b As given by the manufacturer.

There were no statistically significant differences in hemoglobin, hematocrit, platelet count, or fibrinogen between controls and the FVL group at any time points, while MPV was significantly higher for the FVL group at several time points ([Table 2]).

Soluble P-selectin during Pregnancy and Postpartum

sP-selectin concentration was significantly higher in late pregnancy (mean 28 ng/mL) compared with 12 to 14 weeks (20 ng/mL), peaking at delivery (35 ng/mL), and was still increased 2 weeks postpartum (27 ng/mL) compared with 8 to 10 weeks postpartum (20 ng/mL) ([Table 2]). The ratio, sP-selectin/platelet count, also increased significantly in pregnancy, with a peak at delivery and normalization 2 weeks postpartum. The sample with the extremely high platelet count at 2 weeks postpartum had, as expected, a high level of sP-selectin (88 ng/mL) and this result was excluded from statistical calculations. There were no statistically significant differences between controls and the FVL group neither for sP-selectin concentration nor for the ratio sP-selectin/platelet count at any time point. The sP-selectin results for the three women with mild preeclampsia at time of delivery did not deviate from the rest ([Supplementary Fig. S2A], available in the online version only).

Platelet Aggregation during Pregnancy and Postpartum

Platelet aggregation measured by MEA after stimulation with ADP, ASPI, or TRAP followed a U-shaped trajectory, decreasing during pregnancy (ADP 12–14 weeks mean 74 U to 36–38 weeks 44 U, ASPI 97 to 56 U and TRAP 115 to 67 U), with significant lower reactivity for all agonists in weeks 28 to 30 and weeks 36 to 38 compared with 8 to 10 weeks postpartum (P < 0.005) ([Table 2]). Aggregation then increased and exhibited a clear rebound with all three agonists at delivery, before approximate normalization at 2 weeks postpartum. There were no statistically significant differences in platelet aggregation between controls and the FVL group at any time points ([Table 2]). No outliers were detected, and all results were included. The results for the three participants using aspirin during pregnancy (none used postpartum) were included in the overall results as conclusions were not changed if excluded from the statistics, although in gestational weeks 12 to 14 the platelets seemed to be less responsive to addition of ASPI ([Supplementary Table S1A], available in the online version only). Results for the participants using LMWH postpartum (none used in pregnancy) ([Supplementary Table S2A], available in the online version only) and for the three participants with mild preeclampsia at the time of delivery ([Supplementary Fig. S2B–D], available in the online version only) were also included as this did not change the conclusions.

Platelet count (especially <100 × 10⁹/L), hematocrit, and fibrinogen levels are described to impact MEA.[18] [19] [20] [32] As platelet count and hematocrit levels were reduced and fibrinogen was increased in gestational weeks 28 to 30 and 36 to 38 and at delivery, compared with 8 to 10 weeks postpartum, these four time points were chosen for studying the MEA results in relation to platelet count, hematocrit, and fibrinogen ([Supplementary Fig. S3], available in the online version only). No correlation was found between the platelet count and MEA in gestational weeks 28 to 30 and 36 to 38, when the platelet aggregation responses were lowest. At delivery, when the platelet aggregation response started to increase, there was a moderate correlation for platelet count with two of the agonists (ADP and ASPI), and at 8 to 10 weeks postpartum a weak correlation with all three agonists was found. For fibrinogen, a weak negative correlation was observed for one of the agonists (TRAP) in gestational weeks 36 to 38, and a weak positive correlation was found with one of the agonists (ADP) 8 to 10 weeks postpartum. For hematocrit, only a weak negative correlation was found in gestational weeks 36 to 38 with two of the agonists (ASPI and TRAP). After adjusting each platelet aggregation result (ADP, ASPI, and TRAP, respectively) for the hematocrit level in the corresponding sample, the U-shaped trajectory with lowest levels in weeks 28 to 30 and 36 to 38 remained for all agonists ([Supplementary Fig. S4], available in the online version only).

Thromboelastography (TEG) during Pregnancy and Postpartum

Overall, TEG MA, α-angle, and CI were increased throughout pregnancy, whereas R and K times were decreased, compared with 8 to 10 weeks postpartum. Most results were within the reference intervals at 8 to 10 weeks postpartum for all five parameters. LY30 was decreased in late pregnancy and at delivery compared with 8 to 10 weeks postpartum ([Table 3]). Results from women using ASA ([Supplementary Table S1B], available in the online version only) or LMWH ([Supplementary Table S2B], available in the online version only) were comparable to non-users. There were no statistically significant differences between controls and the FVL group for any TEG parameter.

Table 3 Thromboelastography (TEG) variables in pregnancy and postpartum (pp)

Analyte (RI, non-pregnant)	Pregnancy				Postpartum
	12–14 weeks	20–22 weeks	28–30 weeks	36–38 weeks	Delivery	2 weeks pp	8–10 weeks pp
R (4–8 minutes)	Med (2.5–97.5) Mean (−/+ 2 SD)	Med (2.5–97.5) Mean (−/+ 2 SD)	Med (2.5–97.5) Mean (−/+ 2 SD)	Med (2.5–97.5) Mean (−/+ 2 SD)	Med (2.5–97.5) Mean (−/+ 2 SD)	Med (2.5–97.5) Mean (−/+ 2 SD)	Med (2.5–97.5) Mean (−/+ 2 SD)
All, n = 56	5.1 (3.2–7.9) 5.1 (3.1–7.1)[a]	4.8 (2.9–6.6) 4.7 (3.1–6.1)[a]	4.4 (3.4–6.6) 4.6 (3.1–6.1)[a]	4.8 (3.0–7.1) 4.9 (3.3–6.5)[a]	4.1 (2.2–6.1) 4.2 (2.6–5.8)[a]	5.4 (3.6–8.4) 5.5 (3.3–7.7)[a]	5.9 (4.1–9.1) 6.0 (3.7–8.3)
	Med (min; max)	Med (min; max)	Med (min; max)	Med (min; max)	Med (min; max)	Med (min; max)	Med (min; max)
Controls, n = 22	5.2 (3.4; 8.5)	4.8 (2.6; 7.1)	4.4 (3.7; 5.8)	4.8 (2.8; 5.8)	4.0 (2.3; 5.7)	5.4 (3.8; 7.8)	5.8 (4.0; 8.7)
FVL, n = 22	5.2 (3.1; 6.9)	4.7 (3.2; 5.5)	4.7 (3.2; 6.7)	5.2 (3.4; 6.2)	4.2 (2.2; 6.2)	5.3 (3.5; 8.8)	6.0 (4.2; 9.3)
Others, n = 12	4.9 (3.9; 5.8)	5.0 (3.2; 5.8)	4.4 (3.7; 5.7)	4.6 (4.0; 7.5)	4.1 (3.2; 4.8)	5.5 (4.3; 7.8)	5.9 (4.9; 7.4)
K (1–2.1 minutes)
All, n = 56	1.2 (0.8–1.8) 1.3 (0.9–1.7)[a]	1.2 (0.8–1.8) 1.2 (0.8–1.6)[a]	1.2 (0.8–1.7) 1.2 (0.8–1.6)[a]	1.1 (0.8–1.6) 1.2 (0.8–1.6)[a]	1.1 (0.8–1.8) 1.1 (0.7–1.5)[a]	1.2 (0.8–1.9) 1.2 (0.7–1.7)[a]	1.5 (1.0–2.6) 1.5 (0.8–2.2)
Controls, n = 22	1.2 (1.1; 1.6)	1.2 (0.8; 1.6)	1.2 (0.8; 1.7)	1.1 (0.8; 1.5)	1.1 (0.8; 1.6)	1.2 (0.9; 1.7)	1.5 (1.0; 2.9)
FVL, n = 22	1.3 (0.8; 1.8)	1.2 (0.8; 1.8)	1.2 (0.8; 1.6)	1.2 (0.9; 1.6)	1.1 (0.8; 1.4)	1.2 (0.8; 1.9)	1.5 (1.1; 2.1)
Others, n = 12	1.3 (0.8; 1.7)	1.2 (0.9; 1.7)	1.2 (0.8; 1.4)	1.1 (1.0; 1.6)	1.1 (0.8; 1.8)	1.2 (0.8; 1.8)	1.5 (1.2; 1.9)
α-angle (60–73°)
All, n = 56	71 (64–76) 71 (65–77)[a]	72 (65–78) 72 (66–78)[a]	73 (65–78) 72 (66–78)[a]	73 (67–78) 73 (68–78)[a]	74 (66–78) 74 (68–79)[a]	72 (64–77) 72 (65–79)[a]	68 (55–75) 68 (60–76)
Controls, n = 22	72 (65; 74)	73 (69; 77)	73 (65; 76)	73 (69; 79)	75 (67; 78)	72 (65; 76)	68 (53; 75)
FVL, n = 22	71 (63; 77)	72 (64; 78)	72 (66; 76)	72 (67; 77)	73 (68; 77)	72 (63; 77)	68 (59; 73)
Others, n = 12	71 (66; 74)	72 (66; 78)	74 (69; 78)	74 (69; 75)	74 (66; 77)	71 (65; 76)	68 (63; 72)
MA (57–74 mm)
All, n = 56	70 (64–80) 70 (63–77)[a]	70 (59–81) 69 (60–78)[a]	72 (62–79) 71 (63–79)[a]	73 (65–80) 73 (66–80)[a]	73 (62–81) 73 (65–81)[a]	71 (59–81) 71 (62–80)[a]	67 (59–74) 67 (59–74)
Controls, n = 22	69 (65; 74)	69 (59; 75)	72 (61; 76)	74 (64; 79)	74 (65; 80)	70 (58; 81)	65 (59; 73)
FVL, n = 22	70 (64; 79)	70 (61; 80)	70 (64; 76)	73 (68; 80)	73 (66; 81)	70 (65; 77)	67 (59; 74)
Others, n = 12	70 (64; 81)	70 (62; 81)	73 (68; 79)	73 (70; 78)	73 (62; 78)	72 (68; 81)	68 (65; 73)
CI (−3 + 3)
All, n = 56	2.4 (−0.1–4.7) 2.3 (0.3–4.3)[a]	2.6 (0.6–4.8) 2.6 (0.6–4.6)[a]	3.1 (−0.1–6.0) 3.0 (0.6–5.4)[a]	3.1 (0.8–5.3) 3.0 (1.0–5.0)[a]	3.8 (0.8–5.8) 3.6 (1.6–5.6)[a]	2.4 (−0.7–4.4) 2.2 (−0.2–4.6)[a]	1.2 (−2.7–3.4) 1.0 (−1.4–3.4)
Controls, n = 22	2.6 (−0.1; 3.5)	2.5 (0.5; 4.5)	3.1 (0.3; 4.3)	3.2 (0.9; 5.6)	4.0 (1.5; 6.0)	2.2 (−0.8; 4.3)	1.1 (−3.0; 3.6)
FVL, n = 22	2.3 (0; 5.0)	3.0 (0.7; 4.4)	2.7 (−0.3; 6.8)	2.8 (0.9; 4.5)	3.6 (2.1; 5.1)	2.4 (−0.5; 4.4)	1.2 (−2.3; 3.0)
Others, n = 12	2.4 (1.4; 4.2)	2.4 (1.1; 4.9)	3.4 (0.9; 5.0)	3.5 (0.7; 4.0)	3.8 (0.6; 4.8)	2.6 (0; 3.8)	1.2 (0; 2.4)
LY30 (0–5%)
All, n = 56	0.9 (0–3.1) 1.0 (−0.7–2.7)	0.6 (0–3.5) 0.9 (−0.7–2.5)	0.3 (0–2.2) 0.5 (−0.7–1.7)[a]	0.2 (0–1.9) 0.3 (−0.6–1.2)[a]	0.1 (0–2.1) 0.3 (−0.6–1.2)[a]	0.4 (0–2.3) 0.6 (−0.6–1.8)	0.6 (0–2.4) 0.8 (−0.7–2.3)
Controls, n = 22	1.1 (0; 3.3)	0.8 (0; 4.1)	0.3 (0; 2.0)	0.2 (0; 2.1)	0.1 (0; 1.2)	0.4 (0; 2.3)	0.7 (0; 2.4)
FVL, n = 22	0.8 (0; 2.4)	0.6 (0; 2.6)	0.4 (0; 2.3)	0.1 (0; 1.4)	0.2 (0; 1.4)	0.4 (0; 1.8)	0.7 (0; 1.9)
Others, n = 12	0.6 (0; 2.6)	0.6 (0; 1.7)	0 (0; 1.5)	0 (0; 1.1)	0.1 (0; 2.3)	0.3 (0; 2.2)	0.5 (0; 2.4)

Abbreviations: All, all study participants; Angle, angle of clotting (°); CI, coagulation index; Controls, participants without thrombophilia; FVL, participants with heterozygous factor V Leiden mutation; K, time until amplitude 20 mm (min); LY30, percent of lysis at 30 minutes; MA, maximum amplitude (mm); Others, participants with other inherited thrombophilia (heterozygous prothrombin mutation, protein S or C deficiency) or family history of venous thromboembolism; R, time until fibrin formation (min); RI, reference intervals, as given by the manufacturer; SD, standard deviation.

^a Statistically significant differences (p < 0.05) from 8 to 10 weeks pp are marked.

Discussion

No significant differences were observed between women with and without heterozygous FVL in the studied platelet function markers. Although assay sensitivity may play a role, it is plausible that thrombin generation, central to platelet activation, remains comparable between groups in the already hypercoagulable state of pregnancy.[14] [15] The presence of heterozygous FVL may not represent a sufficiently strong risk factor to induce significant changes in platelet aggregation, and the profound pregnancy-related changes in hemostasis may overshadow any subtle effects of FVL. The isolated finding of higher MPV in the FVL group remains unexplained and warrants further investigation.

This study highlights a dynamic and somewhat paradoxical pattern of platelet function across pregnancy. While markers of platelet activation (sP-selectin and TEG MA) increased progressively and peaked at delivery, platelet aggregation measured by MEA followed a U-shaped trajectory: declining steadily through pregnancy, reaching nadir in late gestation, before rebounding sharply within 24 hours postpartum. This transient rebound may reflect acute labor-associated stress, including catecholamine surges, inflammatory cytokine release, and mechanical platelet activation during delivery.[33] The concurrent rise in sP-selectin supports this interpretation and aligns with known physiological stress responses at term.

The observed dissociation between increased platelet activation (sP-selectin, TEG MA) and reduced aggregation capacity (MEA) is notable. One explanation may be that platelets become increasingly activated in vivo during pregnancy, thus increasing sP-selectin, but at the same time rendering these assumed highly activated platelets less responsive to exogenous agonists in vitro, a phenomenon previously suggested by O'Brien et al.[34] Results from Blomqvist et al[35] support the finding of decreased platelet aggregation during pregnancy with ADP, ASPI, and TRAP as agonists, with collagen as an exception. Unfortunately, collagen-induced aggregation could not be assessed in our study due to reagent unavailability. Therefore, we were not able to investigate if the assumed highly activated platelets retain their ability to aggregate in response to collagen as shown in other studies,[35] [36] or if differences between controls and FVL group could be shown.

Of note, the reduced aggregation response was not associated with increased bleeding tendency during pregnancy or delivery (no self-reported increased bleeding tendency or abnormal blood loss among those with the lowest MEA results), suggesting that MEA (ADP, ASPI, TRAP) may not fully capture platelet competence in late pregnancy. Although platelet mass is increased in pregnancy, it is usually insufficient to maintain a non-pregnant platelet count due to hemodilution (plasma volume expansion up to 140%) and accelerated platelet clearance.[37] The rise in MPV during pregnancy may help preserve platelet function despite declining counts, as larger platelets are more reactive and prone to aggregate than smaller platelets.[38]

Platelet count, hematocrit, and fibrinogen concentration may influence platelet aggregation results using MEA.[18] [19] [20] However, platelet aggregation showed overall no significant correlation with these parameters in pregnancy and postpartum, and correcting for hematocrit did not change the U-shaped trajectory. Moreover, platelet aggregation response with MEA started to increase at delivery where hematocrit reached its nadir, indicating that other factors beyond platelet count, hemodilution, and fibrinogen likely contribute to the low platelet aggregation response to agonists in late pregnancy.

The literature is divergent regarding platelet reactivity in pregnancy, but most studies report hyperreactivity using other methods than MEA (e.g., lumiaggregometry[39] [40] and turbidimetry[16]). Discrepancies between studies likely stem from methodological differences, including sample type, anticoagulants, agonists, and timing of assessment.[41] Our findings underscore the complexity of platelet behavior in pregnancy and the limitations of using whole blood aggregometry alone to assess platelet function.

Strengths and Limitations

The foremost strength of the present study was that the same participants were followed longitudinally with standardized sampling procedures and the loss to follow-up rate was low. The results from the study may be used as estimates for normal ranges for MEA and TEG in pregnancy, facilitating interpretation of such tests in clinical practice, although the uncertainty of the reference limits is higher than recommended as fewer than 120 participants were included.

The main limitation was the low number of included women in each group, and some missing samples, which must be taken into consideration when interpreting the data. It was a single-center study with only Caucasians included. This could limit the generalizability to other ethnicities and health care setting. Second was the lack of a biomarker for thrombin generation like F1 + 2 or TAT to verify whether indeed the impact of FVL on thrombin generation is overshadowed by other, pregnancy-related effects, as discussed. Only one method (MEA) for analysis of platelet aggregation was included, and the collagen agonist reagent was not available. TEG MA is affected by platelet activation,[42] but it is also influenced by the increased fibrinogen concentration in pregnancy.[43] Third, a sampling time point 2 weeks postpartum was chosen because of the peaking VTE risk the first few weeks after delivery.[44] Ideally, more time points between 2 and 8 weeks postpartum should have been included to better illustrate the dynamics in this period of increased VTE risk, as well as one reference sample later than 8 to 10 weeks.

Conclusion

During pregnancy, an increase in sP-selectin levels in plasma alongside a decline in platelet aggregation responses to ADP, ASPI, and TRAP measured by MEA, with a rebound postpartum, were found, with no differences in levels between women with and without heterozygous FVL. The lack of significant differences between the groups suggests that presence of heterozygous FVL does not influence platelet activation or aggregation in the context of pregnancy. The observed discrepancy between activation markers and aggregation remains unclear, and further studies using complementary techniques are warranted to clarify the mechanisms underlying the observed trends in markers of platelet function during pregnancy.

Contributors' Statement

M.B.T. and A.H.K.: collected the data; M.B.T.: did the literature research and wrote the first draft of the article; T.H.F.L.: responsible for the analyses of TEG and MEA; M.H.: analysis of sP-selectin; M.B.T., A.H.K., M.H., and C.E.H.: contributed to the statistical work. All authors contributed to the design of the study. All authors contributed to interpretation of data, critical review of the manuscript, and approved the final version of the manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgment

We would like to thank the biomedical scientists at the Laboratory Clinic for excellent technical assistance.

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Publication History

Received: 22 April 2025

Accepted: 18 January 2026

Article published online:
02 February 2026