Abstract
The evolution of the thrombin generation (TG) concept and the recent technical advances in automated TG measurement have made its clinical utility practical but the clinical relevance of all parameters thrombin generation provided still remain unclear. In the current study we investigated effects of various procoagulant plasmatic factors in the TG parameters, such as rate, peak, lag time and area under the curve (AUC), in a platelet poor plasma model system using the calibrated automated thrombography (CAT). The plasma model system was made by mixing well-characterized commercial normal pooled human plasma with plasmas that are deficient in various procoagulant factors (factor VIII, IX, II, X, V and VII) to reach factor levels between <1 % to 100% normal. Initially 5 pM of tissue factor and 4 μM of phospholipid were used and all TG parameters (lag time, peak, rate, and AUC) were compared with various levels of all procoagulant factors. Our result showed that the AUC is proportional to concentrations of factor II and X but not sensitive to factor VIII, IX and VII levels under such experimental conditions. Factor VII affects only the lag time and factor X affects both the lag time and the rate of thrombin generation. Factor II and X affect the peak as well. Factor VIII and IX affect both the rate and peak but not the AUC. Factor V plays very little role on affecting these parameters. Furthermore different concentrations of tissue factor (0.5 to 5 pM) were used to explore the sensitivity of TG for screening hemophiliac factor deficiencies. When ≤ 2pM of tissue factor and 4 μM of phospholipid were used the AUC showed three fold difference for factor VIII deficient plasma versus the normal plasma pool as compared with the less than 20% of difference in AUC (SD=15% for normal population) under the 5 pM concentration of tissue factor. Using the AUC and the rate of TG factor VIII and IX levels were well differentiated under such further diluted tissue factor concentrations (≤ 2 pM). Our study indicated that, under the standard CAT assay condition, 1) the lag time is mainly an indicator of extrinsic tenase reaction (factor VII, X); 2) the peak and rate reflect the intrinsic tenase reaction (factor X, IX, and VIII); and 3) the AUC and peak are largely affected by the prothrombinase factors (II, X) indicating the total free thrombin available for its overall plasmatic procoagulant activity. When proper concentration of triggering agent is used (≤ 2pM of tissue factor and 4 μM of phospholipid) TG becomes sensitive to all plasmatic procoagulant factors and CAT can be used for screening factor deficiencies in hemophiliac patients. A combination of these TG parameters can be used in an in-vitro screening assay to indicate the overall hemostasis balance in plasma under physiological conditions and an individual factor deficiency in related to hemostasis unbalance. Such further diluted triggering agent needs to be optimized for the CAT assay and a standardized large scale clinical retrospective study is needed to prove the utility of these parameters.
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