Coagulation assay with improved specificity to factor V mutants insensitive to activated protein C

Abstract

The prevalence of a new hereditary defect in the protein C anticoagulant pathway, the factor V-Leiden, has been reported to range between 20% to 60% in familial thrombophilia. In addition to differences in patient groups, these very divergent numbers might also be due to the detection method applied. In most studies a modified APTT was used, where activated protein C (APC) is added simultaneously with the start of the clotting reaction. However, this method is also influenced by other factors like protein S, factor VIII or lupus anticoagulants. Furthermore, heparin or oral anticoagulant therapy might interfere. We tried to develop a coagulation assay dependent only on those mutant forms of factor V stable against proteolytic attack by APC. For this purpose, samples were first diluted with a factor V deficient plasma (f.V-dp). Then, coagulation was initiated either on the intrinsic pathway (APTT), or on the extrinsic pathway (PT) or, by directly activating factor X (RVVT). Additionally, APC was added, which prolongation of the clotting time. Deficiencies in protein S or the presence of factor V-Leiden resulted in a less pronounced clotting time prolongation. Titration of protein S-deficient plasma samples with f.V-dp diminished this effect. In contrast, in samples with factor V-Leiden the difference to the clotting time obtained with normal plasma even increased in the order APTT>>RVVT>PT. In the APTT-based method high concentrations of factor VIII shortened the clotting times, thus mimicking a factor V-Leiden defect. This could be compensated for up to 4 U/ml factor VIII by using a f.V-dp containing factor VIII at physiological concentration. Neither unfractionated nor LMW-heparin (up to 2 U/ml) interfered with the determination. In a brief investigation on 16 plasma samples from patients under oral anticoagulation 5 (30%) showed a similar behaviour as observed with normal plasma from factor V-Leiden carriers. These results let us suggest that by simply mixing the patient sample with a factor V-deficient plasma factor V-Leiden might be detected also in patients under oral anticoagulant therapy.