Co-Researcher (RIKEN): Makoto Kaibara Study on Experimental Simulation of Venous Thrombosis Triggering Mechanism Based on the measurements and biochemical analytical results obtained by the rheology measurement system that was developed for in vitro investigation of blood coagulation and thrombus formation under conditions similar to stasis, the possibility of blood coagulation reaction due to the activation of coagulation factor IX by the erythrocyte membrane has been investigated. This study aimed to systematize the blood coagulation reaction mechanism and develop an in vitro experimental simulation in order to investigate the venous thrombosis triggering mechanism. Moreover, basic and practical studies were performed to apply this simulation for the prevention and diagnosis of venous thrombosis. A summary of the studies performed in this project is described as follows: (1) Extraction and identification of factor IX activation enzyme that is expected to be present on the erythrocyte membrane [1] and [2] Based on the investigation of the amino acid sequence of factor IX activation enzyme, it was inferred that this enzyme was similar to elastase that is present on the leukocyte membrane. The in vitro activation of factor IX by this enzyme was approximately 10% of that in the normal blood coagulation reaction. (2) Rheology measurement and analysis of the human blood coagulation process Changes in blood fluidity during the blood coagulation process were measured using a damped oscillation type rheometer in 69 healthy people and patients with various diseases, and the different types of blood coagulation reactions were classified. In approximately 70% of the 69 subjects, blood coagulation was initiated due to the activation of factor IX by the erythrocytes. The initiation time of blood coagulation was controlled by the activation of factor IX by the erythrocytes and the coagulation reaction rate on the erythrocyte membrane after the activation of factor IX. (3) Influence of blood flow on blood coagulation The relationship between the activation of factor IX by erythrocytes and the influence of blood flow on blood coagulation was analyzed by several methods. It became clear that circulatory stasis accelerated the process of blood coagulation remarkably. (4) Analysis of the blood coagulation reaction in different animal species It is important to investigate the occurrence of blood coagulation due to the activation of factor IX by erythrocytes in different animal species for evaluating the application of the model experiment in animals and antithrombogenicity of an artificial heart. It became clear that factor IX activating enzyme did not exist in certain species of animals. (5) Antithrombogenicity of endothelial cells exposed to blood during circulatory stasis [3] Damage to the endothelial cells exposed to blood during circulatory stasis and the resultant blood coagulation were investigated by constructing a hybrid blood vessel model. Since the damage to the endothelial cells did not occur by circulatory stasis alone, it was suggested that the activation of factor IX by erythrocytes played a role in thrombus formation during circulatory stasis. A triggering mechanism Fig. 1 A triggering mechanism of thrombus formation (Activation of factor IX by an enzyme present on the erythrocyte membrane) Fig. 2 Thrombus formation during circulatory stasis (fibrin network) Fig. 3 Antithrombogenicity of endothelial cells exposed to blood during circulatory stasis. (a) Endothelial cells (b) Platelets adherent to endothelial cells Coagulation time and enzymatic activity of erythrocytes Fig. 4 Risk factors for venous thrombosis Fig. 5 Stasis Fig. 6 Age [1] H. Iwata and M.Kaibara: Blood Coagul. Fibrinoly., 13:489, 2002. [2] H. Iwata, M. Kaibara, N.Dohmae, K. Takio, R. Himeno and S. Kawakami: to bepublished. [3] M. Kaibara, T. Yotoriyama and R. Himeno: J. Jpn. Soc. Biorheology, in press.