Activated protein C (APC) resistance

Abstract

Activated protein C (APC) resistance, defined as a poor anticoagulant response of plasma to APC1, was first measured with a modified activated partial throm-boplastin time (APTT) test. This consists of measuring paired clotting times for test plasmas with and without added APC. The extent of prolongation of the clotting time after APC addition over the clotting time before APC addition can be taken as an index of in-vitro APC resistance1. Over the past few years new methods have been developed which are based on the same basic principle, but exploit other tests such as the Russell viper venom (RVV) time2, the prothrombin time (PT)3 and the factor Xa-based (FXa)4. All the above are global clotting tests which explore the coagulation cascade upstream from factor V (FV). Therefore they are, at least in principle, suitable for detection of the FV: Q506 mutation5, and possibly of the specific FV haplotype HR26, which are the most frequent genetic abnormalities associated with APC resistance so far identified. Another method based on the extent of the inactivation of factor VIII (FVIII) measured with chromogenic substrate has been proposed7. More recently an additional method has been described and is based on the measurement of the effect of APC on thrombin generation in plasma upon addition of intrinsic or extrinsic activators of blood coagulation. A common feature of all these methods is their variable sensitivity and specificity with respect to the detection of carriers of the FV: Q506 mutation identified by DNA analysis5, and for some of them it is difficult to interpret results in patients whose basal plasma clotting times are prolonged because of anticoagulant treatment, clotting factor deficiencies9 and the presence of antiphospholipid antibodies10. Dilution of test plasmas in FV-deficient plasma prior to analysis has been proposed to improve the specificity of the APTT11,12 and other methods4 in the above situations. This modification was proven to be effective for patients on oral anticoagulants11,12, but diagnosis in patients with antiphospholipid antibodies still remains a problem, although some methods have been reported to prevent interference 3,13. An additional advantage of using the above modification was shown in a recent large collaborative study designed to compare the diagnostic efficacy of 13 plasma-based clotting methods for their ability to detect the FV: Q506 mutation in comparison with that of the DNA analysis. The results showed that predilution of test plasmas with FV-deficient plasma considerably improved the ability of APTT-based methods to discriminate carriers from non-carriers of the mutation14. However, to make a decision about how to organize a diagnostic work-up in the thrombophilia laboratory, one must also take into consideration that plasma-based methods (namely, APTT-based methods) when performed on undiluted test plasmas are, at least in principle, able to detect acquired APC resistance (i.e. not due to the FV: Q506 mutation), whereas predilution of test plasma in FV-deficient plasma and DNA analysis are not. Whether acquired APC resistance, which has been reported to be frequent in pregnancy15, oral contraceptive intake8,15-17, increased FVIII activity18,19 and cerebrovascular diseases20 is clinically important, and therefore deserves to be detected, is still a matter of debate. Recently, van der Bom et al. 20 have shown in a population-based case-control study that a reduced response to APC, independent of the FV: Q506 mutation, is associated with an increased risk of cerebrovascular disease (the lower the APC response, the higher the odds ratio for cerebrovascular disease). Should this finding be confirmed in subsequent studies and /or extended to venous thromboembolism, graded APC responses measured by APTT-based methods on undiluted plasma could be useful to assess more accurately the thrombotic risk in thrombophilic patients. On the basis of the above considerations, the following section of this chapter will be mainly devoted to the description of the APTT-based method. Additional information for setting up other methods for APC resistance and for DNA analysis for the FV: Q506 mutation can be found in the references cited.