Increased resistance to activated protein C in women taking third-generation oral contraceptives?

The letter by Gris and colleagues raises an interesting point about the interpretation of activated protein C (APC) resistance determined with the endogenous thrombin potential (ETP)–based APC resistance test. Following an earlier debate paper,¹  they argue that the normalized APC sensitivity ratio (nAPC-sr) determined with the ETP-based APC resistance test induces bias of interpretation unless analyzed in relation to the variation of its components, which are the endogenous thrombin potentials (ETP = time integral of thrombin generation) determined in the absence and presence of APC.

Before addressing the question of interpretation, we would like to point out that the nAPC-sr determined with the ETP-based APC resistance test and calculated according the equation used in the paper that we recently published in Blood²  (1) correlates very well with the risk of venous thrombosis for a wide variety of risk factors and combinations of risk factors³  and (2) predicts the occurrence of venous thrombosis in both men and women, also in the absence of the factor V_Leiden mutation, a conclusion that is based on analysis of the Leiden thrombophilia study.⁴ 

The fact that, in the latter study, the predictive value of the ETP-based APC resistance test was the highest in women who were using oral contraceptives (OCs) supports our interpretation regarding the association between changes in APC resistance and the risk of venous thrombosis in women using second- and third-generation OC.

We agree with Gris et al that the variation of the individual components that comprise the nAPC-sr may provide additional information with respect to the mechanistic basis and the thrombotic risk of acquired APC resistance that occurs during OC use. Actually, their argument applies to virtually all functional APC resistance tests, since it is common use in these assays to normalize the hemostatic parameter (clotting time or ETP) determined in the presence of APC through division by the same parameter determined in the absence of APC. Some laboratories subsequently reduce the effect of day-to-day variation on the APC-sr by introducing a “second normalization step” by dividing the APC-sr of patient plasma by the value determined in normal pooled plasma, which yields the so-called normalized APC sensitivity ratio (nAPC-sr).

In retrospect, one may question whether the common practice of expressing APC resistance as an APC sensitivity ratio is a proper action, because this mathematic operation tends to diminish differences observed in the presence of APC (Table 1). In fact, since the protein C pathway is an essential component of the in vivo regulation of thrombin formation, a clotting time or ETP obtained in the presence of APC (ie, with the protein C pathway in action) is likely to be more representative of the physiologic situation and thus may have a higher predictive value for the risk of venous thrombosis than the clotting time (eg, the activated partial thromboplastin time) or ETP determined in the absence of APC. Thus, taking the ratio of clotting time or ETP obtained with and without APC may actually diminish the association with the thrombotic risk.

Therefore, a better measure to evaluate the capability of APC to control coagulation in a given plasma might be the parameter determined in the presence of APC divided by that determined in normal pooled plasma in order to evaluate its deviation from normal. In the case of the ETP-based assay this would result in the following equation: nAPC-sr = ETP + APC (patient plasma)/ETP + APC (normal pooled plasma).

When the data from Table 1 in the letter of Gris et al are used to calculate nAPC-sr according to the “classical method” (nAPC-sr1) or the alternative method described in the previous paragraph (nAPC-sr2), the results presented in our Table 1 are obtained.

Table 1 shows that in both OC users and heterozygous carriers of the factor V_Leiden mutation, nAPC-sr's calculated according the alternative method, in which only the ETPs determined in the presence of APC are taken into account, are actually higher than nAPC-sr's calculated via the classical method. In other words, acquired APC resistance in OC users and the difference between users of second- and third-generation OCs become even more pronounced when only the ETP components determined in the presence of APC are used for evaluation of the anticoagulant effect of APC in plasma.