The Long-Awaited Whole-Blood Thrombin Generation Test

Thrombin is a multifunctional protease that, in addition to hemostasis, supports other “nonhemostatic” mechanisms, such as the regulation of vascular permeability, vascular tone, inflammation, and angiogenesis. There is also evidence that thrombin plays a role in the pathogenesis and progression of liver fibrosis (1). Among the hemostasis functions played by thrombin, one may list the fibrinogen-to-fibrin conversion, the activation of platelets, factors V, VIII, XI, XIII, protein C (PC),2 and the thrombin-activatable fibrinolysis inhibitor. In this issue of Clinical Chemistry, Ninivaggi et al. (2) report on a new whole-blood thrombin generation test (TGT), which has been a long-awaited development in the area of coagulation testing. To understand the importance of this test to the field requires some background information on current coagulation-testing methods. Methods to assess thrombin generation from its precursor prothrombin were developed in the early 1950s, when McFarlane and Biggs triggered coagulation in whole blood or plasma by adding calcium chloride with or without tissue factor or cephalin (3). In the original TGT, the protease generated upon the triggering of coagulation was monitored by sampling a portion of the mixture at fixed time intervals and placing the sample into a second tube containing a fibrinogen solution for testing. The resulting clotting time, which was inversely proportional to the thrombin generated as a function of the coagulation factors, was eventually converted into the thrombin concentration by interpolation from a dose–response calibration curve, which was constructed by testing known amounts of thrombin. The method was too cumbersome and time-consuming for routine use, however, and was soon abandoned in favor of simpler tests, such as the prothrombin time (PT) and the activated partial thromboplastin time (APTT). Many years later, the TGT was revisited by Hemker et al. (4), who introduced important modifications. Fibrinogen as a detection system for thrombin was replaced with a chromogenic substrate specific for thrombin, which allowed the assessment of thrombin by photometric measurement (4). Plasma was defibrinated before testing, thus minimizing the interference produced by the turbidity of fibrin formation during the measurement. Dedicated computer software was developed to calculate the parameter values for the thrombin generation curve, including the lag time for thrombin generation after triggering coagulation, the time to reach the peak, the peak thrombin value, and the area under the curve (referred to as the “endogenous thrombin potential”). The endogenous thrombin potential represents the total amount of thrombin that can be generated as a function of the balance between the procoagulant drivers (coagulation factors downstream from factor XII) and the anticoagulant drivers (namely, antithrombin and PC) operating in plasma. The TGT was further modified by the introduction of a slow-acting chromogenic substrate specific for thrombin that allowed continuous measurement of the cleaved substrate, thereby avoiding the need for the subsample technique. Modified in this way, the method can be fully automated and used on clinical chemistry analyzers (5). More recently, the chromogenic substrate was replaced by a fluorogenic one (6), thus allowing the TGT to be run without defibrination in not only platelet-free but also platelet-rich plasma, a possibility that had not been feasible until then because of the turbidity of the testing mixture. Presently, the TGT can be run on automated fluorometers with use of microplate technology, thereby allowing the simultaneous measurement of thrombin generation for many samples (7).