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SummaryAssays based on the formation of thrombin and fibrin are frequently used, and results are considered exchangeable in research/clinical settings. However, thrombin generation and fibrin formation do not always go hand in hand and flow profoundly influences thrombus formation. We describe the technical/clinical evaluation of an assay to simultaneously measure thrombin generation and fibrin formation under conditions of flow. Introduction of a fluorometer into a ‘cone and base principle’-based rheometer allowed the measurement of thrombin generation (using a thrombin-sensitive substrate) and fibrin formation (changes in viscosity), while applying a linear shear flow. Increasing shear rates inversely related with thrombin generation and fibrin formation. Increasing fibrinogen concentrations in defibrinated plasma resulted in increased thrombin generation and fibrin formation. In pre-operative samples of 70 patients undergoing cardiothoracic surgery, fibrin formation and thrombin generation parameters correlated with fibrinogen content, rotational thromboelastometry (ROTEM) and whole blood Calibrated Automated Thrombinography (CAT) parameters, respectively. Upon dividing patients into two groups based on the median clot strength, a significant difference in perioperative/total blood loss was established. In conclusion, we clinically evaluated a method capable of simultaneously measuring thrombin generation and fibrin formation in plasma/whole blood under continuous flow, rendering our method one step closer to physiology. Importantly, our test proved to be indicative for the amount of blood loss during/after cardiothoracic surgery.

SummaryThe Calibrated Automated Thrombogram (CAT) assay that measures thrombin generation (TG) in platelet-poor and -rich plasma, is increasingly being recognised as a more sensitive tool to determine the overall function of the haemostatic system. We developed a method enabling the measurement of TG in a small aliquot of blood. The objective was to validate this assay in mouse blood and to examine the rate and extent of TG in a mouse model of premature aging. TG was assayed in blood from 20– to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. Bmal1-KO mice are known to display symptoms of premature aging. TG was initiated by adding calcium, tissue factor and a thrombin specific substrate. After TG, the samples were prepared for scanning electron microscopy (SEM). The intra-assay variations (%) in mouse blood of the endogenous thrombin potential (ETP), peak height, lag time, time-to-peak and velocity index were 10% or less (n=24). We found that Bmal1-KO mice have a significantly (p