LAUSR

Why – one might ask – should a methods paper deserve a commentary? Usually, it is when a method is technologically or conceptually novel. That is not the case with the article by Østerud et al. in this issue.1 Rather, this article may help to solve a longstanding problem with the validity of assays of tissue factor (TF) activity. A great number of workers in a wide range of disciplines have developed an interest in the physiological and pathological roles of TF. A particularly active area is the role of TF associated with extracellular vesicles as a marker for a variety of pathologic conditions and a direct contributor to thrombosis.2 There is a great need for a reliable method for assessing TF expression and activity. This is not as easy as it might seem, and many published articles use assays for TF activity that fall short in terms of specificity and sensitivity, calling into question the results of some publications and fueling controversy when different groups report quite different results. The lack of wellvalidated, reproducible, sensitive, and specific assays for TF in blood and other biological samples has hampered progress in understanding the role of TF in human disease and precludes the adoption of TF assays into the clinical laboratory. Of course, one can use an immunoassay to measure the amount of TF antigen. Highly specific antibodies are available for this purpose. However, current immunoassays are not as sensitive as activity assays.3 In many cases, they have insufficient sensitivity to measure the levels of TF that have been associated with a variety of clinical disorders. In addition, in most cases, it is desirable to know whether the TF antigen expresses biochemical activity. It is not entirely straightforward to develop a sensitive and specific activity assay for TF. The enzymology taught in most introductory biochemistry courses is not all that useful when dealing with the specialized proteolytic reactions that make up the hemostatic process. This process is highly regulated, and one mechanism of regulation is that key enzymes (proteases) primarily express their activity when bound to a cofactor on a specialized cellular surface. In addition, all the proteases and some of the cofactors must be activated by proteolytic cleavage before they can exert their functions in hemostasis. Thus, parameters of protease activity depend on the presence and concentration of the cofactor and features of the lipid membrane on which the enzyme, cofactor and substrate are bound.4 This situation makes it difficult to measure the activity of the active enzyme in a physiologically relevant manner. It is harder yet to measure the “activity” of a cofactor, such as TF, which does not itself catalyze a biochemical reaction. Rather, the cofactor influences the kinetic parameters, specificity and localization of its partner protease. Because TF is a cofactor, its activity must be measured indirectly in terms of its ability to modify the catalytic activity of its partner protease, activated factor VII (FVIIa). The activity of a protease can be measured directly by using a smallmolecule chromogenic substrate that is cleaved more or less specifically by the protease of interest to release a colored product. The colored product can then be measured spectrophotometrically, and the development of color is proportional to the activity of the protease. The complex of FVIIa/ TF cleaves two physiologically relevant substrates, coagulation factors IX and X, to their active forms (FIXa and FXa). FIXa is itself difficult to measure because it is not a very good protease in the absence of its own cofactor. Thus, most TF activity assays measure the production of FXa from zymogen FX. Good chromogenic substrates are available for FXa, but prolonged reaction times may be required to measure the low levels of TF activity encountered in biological samples. The sensitivity of the assay can be increased by measuring