Pregnancy Induces a Thrombogenic Platelet Transcriptome in Mice

Background: Venous thromboembolism (VTE) is a leading maternal complication during pregnancy and the immediate post-partum period. Although some studies have documented increasing levels of clotting factors and fibrinolysis inhibitors over the course of pregnancy, little is known about how pregnancy affects platelets. Studies have demonstrated that platelets can be dynamically reprogrammed in response to pathophysiological states such as cancer, infection, and kidney disease, altering their composition, function, and concentration in the circulation. However, the potential of pregnancy to reprogram platelets for an enhanced thrombogenic state has not been investigated.

Aims: We analyzed mouse platelets over the course of pregnancy to identify pregnancy related transcriptomic changes correlated with increased platelet aggregability in response to type 1 collagen and ADP.

Methods: C57BL/6J male and female mice were mated to generate pregnant mice, which was confirmed by the presence of a vaginal plug (embryonic day 0.5). Pregnant mice were randomly assigned to one of four groups based on gestational stages corresponding to human trimesters and the immediate postpartum period (n ≥ 7 per group). The stages were: early (7-9 days post coitus (dpc)), middle (11-14 dpc), late (days 16-20 dpc), and postpartum (days 22-24 dpc). For platelet counts and functional studies, whole mouse blood was drawn via cardiac puncture into a 1:9 volume of buffered sodium citrate. A 20ul aliquot was used for complete blood counts using the Advia 2120. The remaining blood was used for ADP and type 1 collagen platelet aggregation studies using the Roche Multiplate Aggregometer. For the platelet transcriptomic studies, whole blood was drawn via cardiac puncture into acid citrate dextrose/prostaglandin E1. Platelets were isolated via centrifugation and RNA was then extracted and analyzed for purity using the Agilent Bioanalyzer. RNASeq was performed and the data analyzed to identify differences in gene expression conditional upon pregnancy progression. On log2 scale, ≥ 1-fold changes in gene expression were considered to be significantly differentially expressed.

Results: As previously, postpartum platelet counts were significantly increased relative to all groups (control, early, middle and late gestational stages) (p ≤ 0.02, n ≥ 9). We observed a more vigorous platelet aggregation response in the late gestational group compared to the non-pregnant control and early gestational groups (areas under the curve with ADP (p ≤ 0.004, n ≥ 7) and collagen (p ≤ 0.028, n ≥ 7). Transcriptomic analyses revealed nearly 8,000 total mouse platelet transcripts, with just under 700 transcripts statistically significantly altered when comparing the control group to the combined pregnancy and immediate post-partum groups (p≤ 0.05). When comparing each gestational and post-partum groups to control, ≥ 69 transcripts were up-regulated (p≤ 0.05, log change ≥ 1, n ≥ 3). Additionally, when comparing each group to control, there were ≥ 28 down-regulated transcripts (p ≤ 0.05, log change ≤ 1). Initial pathway analyses identified differentially regulated genes with known effects on platelet function, such as glycoprotein 1b-beta (Gp1bb), as well as others whose contribution to platelet function is not well characterized.

Conclusions and future directions: Our results demonstrate that the significant transcriptomic reprogramming of platelets in response to pregnancy are associated with the platelet functional changes observed over the course of pregnancy and the immediate post-partum period. Our results provide the basis for future studies in this area and could lead to improvement in treatment strategies for pregnancy associated VTE.

No relevant conflicts of interest to declare.