Simultaneous measurement of thrombin generation and clot strength in plasma and whole blood

A method for simultaneously determining in real time proteolytic enzyme activity and strength of a fibrin clot formed in a biological sample in a single apparatus, the proteolytic enzyme activity being measured through detection of a signal of a conversion product released upon reaction of a signal substrate with the proteolytic enzyme, the strength of the fibrin clot being measured through a rheometer measuring system, while simulating in vivo blood flow conditions, the method comprising the following steps:

• a. triggering proteolytic enzyme generation in the biological sample so as to enable clotting in said biological sample, and contacting said biological sample with a signal substrate causing a detectable signal related to a conversion product released upon reaction of said signal substrate with the proteolytic enzyme generated, thereby providing a sample mixture,
• b. displaying the sample mixture obtained in step a) as a thin layer in a sample holder, and setting a measuring gap between a rheometer measuring system and said sample mixture, the sample holder being suitable for measurement of a detectable signal obtained after reaction of the signal substrate with the proteolytic enzyme,
• c. applying a rotational force to the sample mixture obtained in step b) through the rheometer measuring system, thereby simulating in vivo venous or arterial blood flow of a determined in vivo physiological or clinical condition,
• d. determining the proteolytic enzyme activity by measuring the development of the detectable signal in the sample mixture obtained in step c) over a period of time and
• e. determining simultaneously the strength of the fibrin clot formed in the sample mixture obtained in step c) by measuring the increase in viscosity of the sample mixture obtained in step c) over a period of time with a rheometer measuring system.