Fluorogenic Assays for Functional Factor VIIa and Tissue Factor.

Blood coagulation is initiated when cryptic tissue factor (TF) becomes exposed on the surface of vascular cells where it can bind circulating factor VIIa (fVIIa). The resulting fVIIa/TF enzyme complex catalyzes the activation of certain blood zymogens that propagate the coagulation event. As a key enzyme for the initiation of coagulation, circulating fVIIa levels have been viewed as an indicator of hemostatic potential and may also be an indicator for the risk of developing cardiovascular disease. The formation of the fVIIa/TF complex is also the basis of specific coagulation assays. Natural or synthetic TF is employed in the prothrombin time (PT assay) where it is used to initiate coagulation in vitro. Therefore, fVIIa and TF play important roles both in vivo and in vitro. Despite such important roles, there have been no simple methods described for measuring the active forms of either protein. This report describes the initial development of an assay for fVIIa and TF that employs the use of an Aminonaphthalenesulfonamide-based (ANSN) fluorogenic substrate. The ANSN substrate is hydrolyzed by fVIIa in a TF-dependent manner, and by titrating fVIIa into a system containing excess TF, the assay becomes sensitive to the fVIIa concentration. Inversely, by titrating TF into a system containing excess fVIIa, the assay becomes sensitive to the TF concentration. This approach has led to the development of both a rapid kinetic and an end-point assay method for each analyte providing detection ranges down to 156 and 1.56 pM respectively. The utility of these assays is displayed by the following applications. Thromboplastin reagents are generally qualified against a W.H.O. standard and are assigned a numerical value known as the International Sensitivity Index (ISI). Theoretically the ISI value should be affected by the quantity and quality of TF in each preparation. An inverse relationship between ISI value and the amount of functional TF would support this theory. To prove this relationship the TF assay was employed to examine two thromboplastin reagents with ISI values of 1.84 and 1.01. The concentration of functional TF measured by this assay was 10.8 nM and 14.6 nM respectively. Because most commercial thromboplastin reagents are prepared by adding TF on a mass basis (as opposed to a functional basis), target ISI values are often missed, leading to an excessive number of failed lots. The TF functional assay provides a method for assigning a specific activity to each TF preparation. By adding TF on a functional basis, lot-to-lot variability would be minimized and target ISI values would be easily achieved. To substantiate this claim, we have determined the functional activity of seven different lots of purified recombinant human TF. With each lot analyzed at a fixed concentration, we observed a broad range of specific activity (range: 8,250 U/sec/mg -14,500 U/sec/mg). These data support the need for a functional TF assay to be incorporated in the manufacturing process of thromboplastin reagents. Lastly, the utility of the fVIIa assay was demonstrated in an experiment using factor VII-deficient plasma samples that were spiked with known quantities of fVIIa. Plasma samples spiked with fVIIa in the range of 0.5-2nM returned mean assay values within 71–100% of the expected values. These data demonstrate the feasibility of using the fVIIa assay to monitor fVIIa levels in hemophilia patients receiving recombinant fVIIa therapy. Further studies will address the circulating concentrations of fVIIa and TF in “normal” and disease state plasma samples.