Summary We have developed a whole blood thrombin generation (TG) assay whereby TG is initiated with a low‐tissue factor concentration and monitored using a fluorogenic thrombin substrate. Significantly higher values were found in blood samples from 50 patients with a history of venous thromboembolism (VTE) compared with 31 healthy controls (HC), for peak height (P = 0·0034) and endogenous thrombin potential (ETP) (P = 0·0027). Results from 31 VTE patients and the 31 controls in the absence of corn trypsin inhibitor (CTI) showed significantly higher values in the VTE group for peak height (P = 0·0013) and ETP (P = 0·002). In the presence of CTI, significantly higher values were only seen in ETP (P = 0·024). No significant increases in TG were found using platelet poor (PPP) or ‐rich (PRP) plasma with or without CTI. The whole blood TG assay in the absence or presence of CTI showed a higher proportion (25/50 and 12/31, respectively) of raised peak height and/or ETP values than plasma assays (PPP 9/50 and 5/31 respectively and PRP 13/50 and 6/31, respectively). Our results show the whole blood TG assay is more sensitive for determining the increases in TG in patients with a history of VTE than PPP and PRP TG assays.

To cite this article: Gallimore MJ, Harris SL, Tappenden KA, Winter M, Jones DW. Urokinase induced fibrinolysis in thromboelastography: a model for studying fibrinolysis and coagulation in whole blood. J Thromb Haemost 2005; 3: 2506-13.Summary. Background: The contact system (CS) proteins, factor XII and prekallikrein are thought to have roles in blood coagulation and fibrinolysis. Recent research has suggested that the CS proteins might be more important in fibrinolysis and cell function than in coagulation. Most studies on fibrinolysis have used plasma or euglobulin assays, ignoring the influence of cellular elements of blood on the fibrinolytic process. Objective and methods: In order to study both coagulation and fibrinolysis in whole blood (WB), we have developed a thromboelastography (TEG) assay to investigate both coagulation and fibrinolysis in the same blood sample. In this assay, named urokinase (UK) induced fibrinolysis in thromboelastography (UKIF-TEG), TEG is performed on recalcified citrated WB in the presence of UK. Large variations in Ly60 (percentage lysis 60 min after clot formation) were obtained between different donors with the same UK concentration. The UKIFTEG assay was therefore performed using UK concentrations that gave Ly60 values in the approximate range of 20-40%. Results: The effect of CS activation was investigated in the presence or absence of celite (10 mg mL )1 blood). Celite shortened the clotting time (CT), and increased Ly60 values. Factor XIIa (FXIIa) and plasma kallikrein (KK) produced concentration dependent reductions in CT (significant at concentrations of 1303 and 2600 ng mL )1 blood, respectively) and increased Ly60 values (significant at concentrations of 652 and 1300 ng mL )1 blood, respectively). Conclusions: Our resultsshow that CS activation and both FXIIa and KK produce reductions in clotting time and enhanced fibrinolysis in UKIFTEG.

We read with interest the comments by Al Dieri and Hemker on our recent article on thrombin generation (TG) in plateletpoor plasma (PPP), platelet-rich plasma (PRP) and whole blood (WB) (Tappenden et al, 2007).Al Dieri and Hemker report that they have tried unsuccessfully to reproduce our method for determining TG in WB. They suggest that, because of this, we must have omitted one or more details of the experimental procedures in our paper. They base their argument on two major points.Their first point deals with the fluorescent signals obtained in WB. They state that they found these to be 30· weaker in WB than those in plasma and that the signal to noise ratio was so poor in WB that they were unable to obtain useful first derivatives.The results described in our paper follow over 2 years development work on the plasma-based and WB TG methods including comparisons of different microtitre plates (clear or black and shape), studies on a variety of fluorogenic peptide substrates, the determination of the actual amounts of tissue factor added in the assays and excitation and emission wavelength scans to find optimal assay conditions. These studies were performed using a Gemini XS fluorimeter (Molecular Devices, West Sussex, UK), which is equipped with a monochromator rather than a filter wheel and has the ability to mix the incubating material between readings. In our method the samples were read every 30 s and continually mixed during the incubation period between readings. A major influence on obtaining results in WB was the use of different excitation and emission wavelengths for WB compared to those used for PPP and PRP. Using the wavelengths specified the signal obtained for WB was two to three times weaker than that seen in plasma samples. We were able to obtain relative fluorescence units (RFUs) in excess of 3500 in WB. This produced a signal to noise ratio (at an RFU of 2600) some 3· poorer than that obtained in PPP. This did not however prevent the calculation of the first derivative.The second point in the letter by Hemker and Al Dieri deals with erratic TG curves obtained with WB and the conversion of the fluorescence data. While the curves obtained in the TG assay are more erratic in WB than those in PPP (Fig 1A and B) they still allow for conversion to the first derivative and thus the calculation of the thrombin concentration. In those samples where the fluorescence is higher than that of the calibrator, the effect may be an underestimation of the inner filter effect; this may reduce the final thrombin concentration calculated by the mathematical model. It is worth noting that in comments made by AlDieri and Hemker they fail to state the type of fluorimeter used in their experiments. We believe the reason for our success in measuring TG in WB is not only because of the optimized assay conditions and wavelengths chosen, but also to the type of fluorimeter used. We surmise that this is probably as a result of the fluorimeter using a monochromator rather than a filter wheel but there may be other propert...