Thrombomodulin is a stronger indicator of combined oral contraceptives-induced activated protein C pathway resistance in the thrombin generation test than activated protein C

Abstract

Background: The mechanism by which combined oral contraceptives (COCs) lead to hypercoagulation is not fully understood, although activated protein C (APC) pathway resistance has been implicated. APC and thrombomodulin (TM) tend to be considered as interchangeable reagents, even though their biological action in coagulation is different. However, it remains unclear which reagent is better suited for the detection of APC pathway resistance. We compared the effectiveness of TM and APC in TG to detect COC-induced APC pathway resistance using thrombin generation (TG).

Methods: TG was measured on ST Genesia in 48 healthy women, of whom 24 used COCs. TG was triggered with STG-ThromboScreen (with and without TM), spiked with a low and high concentration of TM or APC (2 or 15 nM TM, or 1.5 or 5.5 nM APC), aimed to achieve 50% and 90% ETP inhibition, respectively.

Results: TG was higher in women using COCs. TM and APC inhibit TG in all women, although their inhibitory effect is more pronounced in women without COC compared to women with COC. The addition of 2 nM TM causes an ETP reduction of 40% (1,289 vs. 768 nM•min) in women without COC and an ETP reduction of 24% (1,704 vs. 1,287 nM•min) in women with COC. The addition of 1.5 nM APC causes an ETP reduction of 41% (1,289 vs. 759 nM•min) in women without COC and an ETP reduction of 23% (1,704 vs. 1,316 nM•min) in women with COC. The difference in effect between women with and without COC is largest when 15 nM TM, aimed at 90% ETP inhibition, is used. 15 nM TM leads to the smallest overlap in ETP inhibition between women with and without COC (27% overlap), compared to 2 nM TM (41% overlap), and 1.5 nM APC (38% overlap) and 5.5 nM APC (41% overlap).

Conclusion: Although TM and APC are often used interchangeably to assess the sensitivity of the APC system in TG, our findings suggest that TM is a better discriminator to detect COC-use induced APC pathway resistance. In addition, we found that the ETP is a better TG test readout for APC pathway resistance testing than the peak height.

Keywords: activated protein c; coagulation; combined oral contraceptives; thrombin; thrombomodulin.

Figure 1

Average TG curves in women with and without combined oral contraceptives and the effect of added TM and APC. (A) TG curves in women without COC (continuous line) and women with COC (dashed line). (B-C) Effect of Thromboscreen TM, 2 nM and 15 nM in-house TM, and 1.5 nM and 5.5 nM in-house APC in women without COC (B) and women with COC (C).

Figure 2

The effect of TM and APC on TG parameters in women with and without combined oral contraceptives. (A) TG lag time was comparable in women with and without COC. (B,C) The effect of TM and APC on lag time in women without COC (B), and with COC (C). (D) Peak height was significantly higher with COC compared to women without COC. (E-F) The effect of TM and APC on peak height in women without (E) and with COC (F). (G) Time-to-peak was significantly shorter in women with COC compared to women without COC. (H,I) The effect of TM and APC on time-to-peak in women without COC (H) and women with COC (I). (J) ETP was significantly higher in women with COC compared to women without COC. (K,L) The effect of TM and APC on ETP in women without COC (K) and women with COC (L). (M) Velocity index was significantly higher in women with COC compared to women without COC. (N,O) The effect of TM and APC on velocity index in women without COC (N) and women with COC (O). Data are shown as individual values with mean and standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. APC, activated protein C; ETP, endogenous thrombin potential; TM, thrombomodulin.

Figure 3

The dose-dependent effect of in-house TM and APC on ETP and peak height. (A) The absolute inhibitory effect of 2 and 15 nM TM on ETP in women with and without COC. (B) The absolute inhibitory effect of 1.5 and 5.5 nM APC on ETP in women with and without COC. (C) The absolute inhibition of the ETP by TM and APC. (D) The relative inhibitory effect of 2 and 15 nM TM on ETP in women with and without COC. (E) The relative inhibitory effect of 1.5 and 5.5 nM APC on ETP in women with and without COC. (F) The percentage inhibition of the ETP by TM and APC. (G) The absolute inhibitory effect of 2 and 15 nM TM on peak height in women with and without COC. (H) The absolute inhibitory effect of 1.5 and 5.5 nM APC on peak height in women with and without COC. (I) The absolute inhibition of the peak height by TM and APC. (J) The relative inhibitory effect of 2 and 15 nM TM on peak height in women with and without COC. (K) The relative inhibitory effect of 1.5 and 5.5 nM APC on peak height in women with and without COC. (L) The percentage inhibition of the peak height by TM and APC. Data are shown as (individual values with) mean and standard deviation. *p < 0.05, **p < 0.001, ***p < 0.001, ***p < 0.0001. APC, activated protein C; ETP, endogenous thrombin potential; COC, combined oral contraceptives; TM, thrombomodulin.

Figure 4

Comparison of the effect size of TM and APC addition for the discrimination between women with and without COC. (A) Effect size of the inhibition of ETP in women with vs. without COC when using TM or APC. (B) Effect size of the inhibition of peak height in women with vs. without COC when using TM or APC. Data are shown as the Glass's delta statistic for each reagent when comparing COC- and COC + women. The grey area in the graph indicates Glass's delta statistic values that are considered to be a large effect. APC, activated protein C; ETP, endogenous thrombin potential; TM, thrombomodulin.