Studies on the initiation of extrinsic blood coagulation reaction

为了了解人组织因子胞外结构域的功能差异，我们表达了可溶性形式的组织因子突变体，其携带凝血因子Xa的易感位点以产生N-和C-结构域。重组组织因子突变体在酵母系统中表达，并通过柱层析从培养基中纯化。分离的N-结构域没有显示出任何凝血因子VIIa的加速活性。然而，C-结构域显示完整组织因子的5%活性。我们建立了用重组可溶性组织因子测定血浆活化因子VII（VIIa）水平的方法。我们在1 ml人血浆中鉴定出2-4 ng VIIa，相当于总因子VII水平的0.5-1%。我们观察到心肌梗死、脑梗死和糖尿病患者血浆VIIa水平较高。微量白蛋白尿的糖尿病患者显示更高的VIIa水平。VIIa水平与内皮细胞损伤标志物von Willebrand因子相关。因此，我们发现，糖尿病患者显示高血浆因子VIIa水平伴随着内皮损伤。

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.