The Activated Protein C (APC) Response to In vivo Thrombin Formation Correlates with the Thrombotic Risk in Factor V Leiden but not in Prothrombin G20210A Carriers

Objectives: Factor V Leiden (FVL) and prothrombin G20210A (PRO) are the most frequent genetic risk factors for venous thromboembolism (VTE). Recently, we have shown increased generation of activated protein C (APC) in asymptomatic FVL carriers (VTE-) in response to thrombin formation, induced by recombinant activated factor VII (rFVIIa) in vivo (Rühl et al. Blood 2018;131:1489–1492). Aim of the present study was to extend this approach to carriers of PRO and FVL with prior VTE (VTE+).

Methods: Carriers of FVL (29, 25 heterozygous, hz: 13 VTE-, 12 VTE+) and PRO (26, 24 hz: 12 VTE-, 12 VTE+) as well as 13 healthy non-carriers were included. No subject was under anticoagulant treatment at time of analysis. Blood samples were collected immediately before and during 8 hours after injection of 15 µg/kg rFVIIa. Thrombin and APC were quantified using oligonucleotide-based enzyme capture assays (OECAs). Prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complexes (TAT), and D-dimer were measured additionally.

Results: rFVIIa was well-tolerated by all subjects and median D-dimer levels remained within the reference range in all groups. Compared with the healthy controls, peak plasma levels of F1+2 and TAT were higher after rFVIIa injection in FVL (p = 0.009 and p = 10⁻⁴), and in PRO carriers (p = 0.010 and p = 2·10⁻⁴). They did not differ between FVL and PRO, and between VTE+ and VTE- subgroups. Median thrombin levels were below the limit of detection at baseline and remained unchanged after rFVIIa injection. APC increased from 0.63 (0.54–1.16) to 3.14 (2.55–3.55) pmol/L (p = 0.017) in the controls, and from 1.39 (1.14–1.86) to 7.86 (5.55–11.55) pmol/L (p < 10⁻⁴) in the FVL group. In PRO carriers, APC increased from 1.03 (0.59–1.17) pmol/L to 5.73 (4.00–6.57) pmol/L (p = 7·10⁻⁴). Peak plasma levels of APC were higher in carriers of FVL and PRO in comparison to controls (p < 10⁻⁴ and p = 4∙10⁻⁴, respectively), and lower in PRO than in FVL carriers (p = 0.002). With peak levels of 8.11 (7.59–11.77) versus 5.62 (4.94–6.49) pmol/L, APC was significantly higher in the VTE- than in the VTE+ group (p = 0.004) in hz FVL carriers, while in hz PRO carriers, APC peak levels did not differ between VTE- and VTE+ ([Fig. 1]).

Conclusions: The APC response to in vivo thrombin formation is significantly higher in asymptomatic FVL carriers than in those with a history of VTE, implying that higher APC levels protect FVL carriers from thrombosis development. This hypothesized effect appears to be specific for FVL as PRO carriers do not show differences in the APC response related to VTE history. Further studies are warranted to identify the factors that modulate the APC response in FVL carriers.

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