The multiple faces of the partial thromboplastin time APTT

Abstract

In the historical sketch by G. C. White [1], the importance of the activated partial thromboplastin time (APTT) for research on coagulation and hemophilia is emphasized. I wish to add that the APTT method has also had a major impact on research on the anticoagulant pathways and on thrombophilia. In this brief comment, I reflect upon the role played by the APTT in the discovery of activated protein C (APC) resistance and on the importance of the method for the elucidation of the underlying molecular mechanisms of this condition. The observation initiating the work on APC resistance was made while performing a functional protein C assay on plasma from a patient with thrombosis [2]. The protein C assay included isolation of protein C by barium citrate adsorption, activation of the protein C by a thrombin–thrombomodulin complex and finally an APTT reaction to monitor the anticoagulant response of APC. In the analysis, it was observed that the patient sample and the standard did not yield parallel dose–response curves. This stimulated our curiosity and we performed a large number of experiments to understand the problem. During these studies it became apparent that the unexpected behavior was not related to the patient's protein C but that something was wrong with the patient's plasma—we found a poor anticoagulant response of the patient's plasma to added normal APC. This observation lead to the design of a new APTT-based assay in which normal APC was added to the patient's plasma and the anticoagulation response measured. Using this first version of the APC resistance test, we found that not only the original patient but also many of his family members responded poorly to APC. Stimulated by these observations, we and others investigated cohorts of thrombosis patients and it became obvious that APC resistance was a major risk factor for venous thrombosis [3-5]. Moreover, in a majority of the cases, the APC resistance was inherited and was due to the same genetic defect [4, 6]. The APTT-based APC-resistance test was instrumental in identifying factor (F)V as the causative molecule for the APC-resistance phenotype and laid the foundation for the subsequent identification of the responsible mutation in the FV gene [7]. Almost all patients with APC resistance were found to carry the same mutation, which replaces Arg506 in one of the APC-cleavage sites with a Gln (FV Leiden) [8-11]. As a consequence of this mutation, the APC-mediated degradation of FVa was impaired. This observation has provided great stimulus to investigations of the mechanisms of APC-mediated degradation of FVa and factor (F)VIIIa and the role of protein S. During the efforts to elucidate the molecular mechanisms of APC resistance, it was discovered that FV is a Janus-faced protein with a potential to express both pro- and anticoagulant activities [7, 12, 13]. The anticoagulant function of FV is that of a cofactor to APC in the degradation of FVIIIa. In APC resistance, this anticoagulant property of FV is lost due to the FV Leiden mutation. The first diagnostic assay for APC resistance, the ‘classical’ APC-resistance test, was modified based on the discovery of the involvement of FV in the pathogenesis of APC resistance. In the modified test, FV-deficient plasma was included in the clotting reaction [14]. The modified APC-resistance test has been found to be highly specific and sensitive for the FV Leiden mutation and is often used as a first screening method to identify individuals with APC resistance. One might reflect on what made the APTT reaction particularly suitable for research on APC resistance. A contributing factor is that the APTT is dependent on the activity of both FVa and FVIIIa. It is therefore sensitive to the effect of APC at two levels of the coagulation reaction. In contrast, the regular prothrombin time reaction is sensitive to changes only in FVa activity and unlike the APTT it is sensitive neither to loss of FVIIIa activity nor to loss of the anticoagulant function of FV. In conclusion, I wish to add to the praise of the APTT method. It was important not only for defining a new era in coagulation but also for research on anticoagulant pathways, for the discovery of APC resistance and the elucidation of pathogenic mechanisms of thrombophilia.