A chromogenic assay for activated protein C resistance.

Resistance to activated protein C (APC) diagnosed on the basis of prolongation of clotting time in an activated partial thromboplastin time (aPTT) assay is now considered a major cause of inherited thrombophilia. The majority of patients with APC resistance carry a factor V molecule with a point mutation at one APC cleavage site (Arg506Gln) which prevents the optimal inactivation of activated factor V by APC. To overcome the limitations of aPTT-based assays in the diagnosis of APC resistance, we have developed a chromogenic assay which is based on the capacity of APC to limit the generation of factor Xa by inactivating factor VIIIa in plasma. The ratio of the factor Xa amidolytic activity in a sample without APC to its factor Xa activity with the addition of APC reflects the response of the plasma coagulation system to APC. The normal range in 44 healthy individuals was 1.62-2.06. APC response ratios as measured by the chromogenic assay correlated with ratios measured by the aPTT assay and were below the normal range in 23/24 individuals with Arg506Gln mutant factor V from three different families with familial thrombosis and from 11 unrelated asymptomatic individuals. In reconstitution experiments, purified factor V corrected the decreased APC response in plasma samples from patients with the Arg506Gln mutation as well as with factor V deficiency, and increased the APC response in normal plasma, whereas the addition of activated factor V had no enhancing effect.