A CHROMOGENIC ASSAY FOR ACTIVATED PROTEIN-C RESISTANCE

Resistance to activated protein C (APC) diagnosed on the basis of prol ongation of clotting time in an activated partial thromboplastin time (aPTT) assay is now considered a major cause of inherited thrombophili a. The majority of patients with APC resistance carry a factor V molec ule with a point mutation at one APC cleavage site (Arg506Gln) which p revents the optimal inactivation of activated factor V by APC. To over come the limitations of aPTT-based assays in the diagnosis of APC resi stance, we have developed a chromogenic assay which is based on the ca pacity of APC to limit the generation of factor Xa by inactivating fac tor 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 A PC reflects the response of the plasma coagulation system to APC. The normal range in 44 healthy individuals was 1.62-2.06. APC response rat ios as measured by the chromogenic assay correlated with ratios measur ed by the aPTT assay and were below the normal range in 23/24 individu als 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 mutat ion as well as with factor V deficiency, and increased the APC respons e in normal plasma, whereas the addition of activated factor V had no enhancing effect.