A New Functional Coagulation Assay for Measurement of Total Factor V Activity in Human Plasma and Its Application to Samples from Patients with Disseminated Intravascular Coagulation.

Abstract

Factor V (F.V) circulates as an inactive single chain procofactor. Only upon cleavage and activation with thrombin to F.Va does it function as a cofactor in the F.Xa-mediated conversion of prothrombin to thrombin. The conventional F.V coagulation assay measures the one-stage clotting activity, which most likely reflects the trace of F.V that is activated in the sample or becomes activated during the assay. Since F.V is activated by thrombin to F.Va, a second (two-stage) assay is crucial to fully assess the total F.V potential activity in the plasma sample. For this assay, normal human pooled plasma (diluted 100- to 500-fold) was treated with thrombin (1–5 NIH Units/ml, final concentration) for various times at 37°C to fully activate the F.V. The added thrombin was inhibited with a 2-fold molar excess of Phe-Pro-Arg chloromethylketone (PPACK) and the sample re-assayed. This second assay measures the total F.V activity of a sample. Maximal 10-fold activation of plasma F.V activity occurred with 2Units/ml thrombin after 1 to 2min at 37°C and this activity was stable for 30min prior to PPACK addition. Control experiments indicated that the levels of PPACK used did not affect the one-stage F.V activity of untreated plasma samples but were required to inhibit the added thrombin from clotting fibrinogen in the assay. Assay of timed plasma samples (n=48) from seven patients admitted to the Intensive Care Unit and subsequently diagnosed with disseminated intravascular coagulation (DIC), indicated that the total F.V activity, the specific activity (total F.V activity/antigen), and the activation quotient were decreased, on average, by 45%, 49%, and 31%, respectively, from that observed with normal human pooled plasma. The results indicate that the total amount of activatable F.V as well as its degree of activation decreases from normal as part of the pathogenesis of DIC in humans. Further, the decreased total F.V specific activity observed in DIC patient plasma samples indicates that the F.V that is present is less active per molecule than normal, possibly because of proteolytic inactivation. This total F.V clotting assay should provide new information about the biochemistry of F.V and its contribution to the pathogenesis of coagulation disorders.