Measuring the mechanical properties of blood clots formed via the tissue factor pathway of coagulation

Abstract

Thrombelastography (TEG) is a method that is used to conduct global assays that monitor fibrin formation and fibrinolysis and platelet aggregation in whole blood. The purpose of this study was to use a well-characterized tissue factor (Tf) reagent and contact pathway inhibitor (corn trypsin inhibitor, CTI) to develop a reproducible thrombelastography assay. In this study, blood was collected from 5 male subjects (three times). Clot formation was initiated in whole blood with 5 pM Tf in the presence of CTI, and fibrinolysis was induced by adding tissue plasminogen activator (tPA). Changes in viscoelasticity were then monitored by TEG. In quality control assays, our Tf reagent, when used at 5 pM, induced coagulation in whole blood in 3.93 ± 0.23 min and in plasma in 5.12 ± 0.23 min (n=3). In TEG assays, tPA significantly decreased clot strength (maximum amplitude, MA) in all individuals but had no effect on clot time (R time). The intraassay variability (CVa<10%) for R time, angle, and MA suggests that these parameters reliably describe the dynamics of fibrin formation and degradation in whole blood. Our Tf reagent reproducibly induces coagulation, making it an ideal tool to quantify the processes that contribute to mechanical clot strength in whole blood.

Figure 1

Tissue factor quality control. Whole blood (panel A) and plasma (panel B) clot times increase dose dependently as the Tf concentration is decreased from 10 pM to 2 pM. Symbols (○, ●, ▼) represent data generated from 3 independent preparations of Tf. At a nominal concentration of 5 pM Tf which corresponds to the concentration used in out TEG assay, the whole blood and plasma clot times are 3.93 ± 0.23 and 5.12 ± 0.23 minutes, respectively (panel C).

Figure 2

TEG tracings show the individualized response to tPA. Clot-lysis profiles are depicted for all 5 subjects in the absence of tPA (solid line) and with 1 (long dash), 1.5 (short dash) or 2 nM tPA (dash dot). The data represent the mean ±SD, n=3 blood draws (each experiment performed in duplicate).