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May 1, 2012 (Vol. 32, No. 9)

Thrombin Generation Assay Aids Development

Calibrated Automated Thrombogram System for Global Hemostasis Measurements

  • Historically, hemostasis has been divided into three parts: (1) blood vessel constriction, (2) primary hemostasis resulting in a platelet plug, and (3) plasmatic-based blood coagulation. However, in the past decade it has become clearer that primary hemostasis and plasmatic hemostasis are so tightly interwoven that this perceived distinction is relatively artificial.

    At the site of the injury, tissue factor (TF), collagen, and cell debris are exposed to the blood flow leading to platelet attachment and activation, with thrombin playing a central role in the process after the initial events of TF/factor VIIa-mediated activation.

    In addition, thrombin activates additional clotting factors and platelets in order that more thrombin formation will be accelerated. The platelets that are adhered to the site of the wound are tied together by fibrin threads formed after activation of fibrinogen by thrombin. Laboratory testing of platelet activation and blood coagulation helps to understand the interplay between these various pathways and how an experimental treatment affects them on an individual, multifactor, or global level.

    There are many tests available for function testing of the various steps and individual factors in the hemostasis process. However, global hemostatic capacity testing, through the use of thromboelastography or thrombin generation assays examining the blood coagulation pathway, has been heavily used for several decades in order to characterize various components of the coagulation pathways.

  • Biology of Hemostasis

    The initial step after tissue injury in the body is rapid constriction of the blood vessels. The second step is formation of the platelet plug via interaction between multimerized von Willebrand’s factor and platelets. The third step is a rapid acceleration of the extrinsic pathway of blood coagulation.

    TF release results in the formation of thrombin, which then activates fibrinogen to fibrin, resulting in polymerization of fibrin and formation of the fibrin clot. Though formation of the fibrin clot is the final step in this process, arresting blood loss physiologically, thrombin is the centrally regulated enzyme in this process.

    If one wished to view the complex process from a global perspective, what step would be examined? Though vessel constriction is a necessary first step in the process, it alone does not cause clotting to stop completely, evidenced by the fact that hemophiliacs have normal vessel constriction function but still bleed, sometimes severely. Similarly, patients with defects of von Willebrand’s factor or platelet function will still bleed, sometimes in a severe manner.

    From these clinical observations, the focus of researchers wishing to investigate global hemostatic function has usually been around intensive study of plasmatic-based coagulation. In the coagulation system, global hemostatic tests have either been centered on measurement of the fibrin clot (thromboelastography) or on thrombin’s enzymatic activity, since these are the two furthest downstream events in the system.

  • CAT Assay: Principle, Protocol, Instrumentation

    Click Image To Enlarge +
    Figure 1. The instrument on which the Calibrated Automated Thrombogram (CAT) test is run as a semi-automated thrombin generation method.

    Thrombin generation (TG) works by introducing a thrombin-specific chromogenic or fluorescent substrate to clotting plasma, permitting measurement of the activity of thrombin as a function of time. The Calibrated Automated Thrombogram (CAT) assay developed by Thrombinoscope/Diagnostica Stago is a research use only semi-automated method for running TG assays.

    Testing on the CAT begins by first placing normal and treated plasmas of interest into six wells of a 96-well plate, followed by introduction of TF or the thrombin calibrator to three of the six total wells. The patented thrombin calibrator allows for correction of substrate consumption and nonlinearity of the test as well as donor-to-donor color differences of the plasma, turbidity, or hemolysis.

    After the plate is filled with all platelet-poor or platelet-rich plasmas of interest, it is placed into a Thermo Fluoroskan Ascent 96- well fluorescent plate reader from Thermo Scientific (Figure 1). A dispenser inside the instrument then dispenses the fluorescent substrate/Calcium mixture into each well and the fluorescence from the plate read for up to 60 minutes.

    After the run is finished, software specific to this application will then calculate all pertinent parameters in the thrombogram curve in order to assess the relative activity of the plasma being tested. The parameters include the lag time, time to peak, area under the curve (also known as the endogenous thrombin potential, ETP), slope of thrombin formation (also known as the velocity index, VI), and the time to peak completion (Figure 2).

    Past standardization studies have shown that when the ETP of the plasma being tested is compared to normal plasma run on the same plate, the researcher can fully understand how the bulk coagulation activity of the plasmatic coagulation system has been affected.

  • Comparison of Global Hemostasis Assays to other Methods

    Click Image To Enlarge +
    Figure 2. The thrombogram readout is shown with associated parameters displayed for a normal recalcified plasma sample activated by a standard concentration of recombinant tissue factor to initiate coagulation.

    Widely-used coagulation tests include the prothrombin time (PT) and the activated partial thromboplastin time (aPTT). While the PT and aPTT assays have the strong benefit of being automation friendly and well standardized, they are not sensitive to minor changes in the thrombogenicity of the hemostatic system.

    In particular, the lag time shown in Figure 2 corresponds to the amount of thrombin formed during the PT, which as shown is a very small proportion of the total thrombin capacity of the test plasma.

    In comparing the CAT to other TG-based tests, tests including those utilizing a chromogenic substrate are interfered with by formation of the fibrin clot, resulting in the requirement that the fibrin clot is inhibited, which changes the physiologic dynamics of clot formation.

    In addition, platelets cannot be tested in the chromogenic assay system due to their light-interfering properties. Other TG assays (available commercially or laboratory developed) may not include the thrombin calibrator, making them vulnerable to varying degrees of bilirubin, lipemia, and hemolysis present in different donor plasmas.

    In addition, substrate consumption is critical to correct for, especially if the test plasma treatment directly affects the activity of thrombin. The choice of TF concentration is also critical in order to make the system as sensitive as possible to minor coagulation perturbations. Other methods for TG and thromboelastography may contain very high TF concentrations, making them less sensitive overall.

  • Use of TG and CAT

    In addition to academic research needs, measurement of TG is a critical need for commercial research labs wishing to develop and validate new anticoagulant or antiplatelet drugs. In one publication, the differential effects of inhibitors of factor Xa (a different component of the blood coagulation system) and thrombin were determined. In addition, TG testing is useful for validating intravenous immunoglobulin preparations for the presence of procoagulant contaminants.

  • Summary

    In summary, TG testing using the published CAT assay allows researchers to run TG tests in a well-controlled, standardized, semi-automated, and medium-throughput fashion in the basic research laboratory. The CAT method, by providing an accessible method to understand in bulk the coagulability of a plasma sample, helps to illuminate this otherwise complex pathway.

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