Automated fluorogenic methods for the evaluation of the extrinsic coagulation reactions in human plasma

Highly sensitive automated methods for the evaluation of the extrinsic coagulation reactions in human plasma were developed by the combination of fluorogenic peptide substrate (MCA) for thrombin and a centrifugal autoanalyzer (Cobas Bio). Prothrombin time (PT) was measured by the reaction time to reach 0.1 relative fluorescence which was caused by the action of thrombin generated after the activation of 3 microliters plasma with human placental tissue factor (Thromborel S). Factors X and VII contents in plasma were measured by the same method after mixing diluted plasma with each factor deficient plasma, tissue factor, calcium and MCA in which 10-800% of each factor was quantitatively measured. Prothrombin content in plasma was quantitated by measuring thrombin activity after the activation with human activated Factor X in the presence of phospholipid and calcium in which 10-160% of prothrombin was measured. By the application of these fluorogenic methods to the patients with cardiovascular diseases, it was demonstrated that these methods are highly sensitive not only to hypocoagulable state, but also to hypercoagulable state, particularly to the increase of Factors X and VII concentrations in plasma.