Studies on the initiation of extrinsic blood coagulation reaction

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To understand the functional discrimination of extracellular domains of human tissue factor, we have expressed soluble form of tissue factor mutant which carries the susceptible site of coagulation factor Xa to make N- and C-domains. The recombinant tissue factor mutant was expressed in yeast system and purified from the culture medium by using column chromatography. The isolated N-domain did not show any acceleration activity of coagulation factor VIIa. The C-domain, however, showed 5% activity of the intact tissue factor. The combination of N- and C-domains restored the activity at the original level, indicating that the both domains of tissue factor are reguired for the activity.We have established the assay methos to measure the plasma level of activated factor VII (VIIa) using the recombinant soluble tissue factor. We identified 2-4 ng VIIa in 1 ml of human plasma, that corresponds to 0.5-1% of total factor VII level. We observed the higher plasma VIIa levels in patients with myocardial infarction, cerebral infarction, and diabetes. The diabetes patients with microalbuminuria showed much higher VIIa levels. The VIIa levels were correlated with endothelial cell damage marker, von Willebrand factor. Thus, we showed that the diabetes patients showed high plasma factor VIIa levels concomitant with the endothelial damage.