2؉ion, which formed a bridge between IXGD1-46 and IXbp, forced IXGD1-46 to rotate 4°relative to IX-bp and hence might be the cause of a more tight interaction between the molecules than in the case of the Mg 2؉ -free structure. The results clearly suggest that Mg 2؉ ions are required to maintain native conformation and in vivo function of factor IX Gla domain during blood coagulation.

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Prothrombin is expressed in the central and peripheral nervous systems. However, the mechanism responsible for the activation of prothrombin to thrombin by the activated form of factor X in the central and peripheral nervous systems remains to be explored. Here, we investigated the expression of factor X mRNA in the brain and some cell lines derived from the central nervous system. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated the expression of mRNA encoding factor X in the rat brain, A172 (human glioblastoma) and GOTO (human neuroblastoma) cells. The sequences of PCR-derived fragments were identical to those reported for rat and human factor X. These results indicated the synthesis of factor X in the cells of the central nervous system. z 1999 Federation of European Biochemical Societies.

A potent antidote-controlled aptamer, as an anticoagulant, has reportedly overcome the complications of acute bleeding by the administration of available anticoagulants. In the present study, we provide evidence that the aptamer binds specifically to factors IX and IXa and inhibits their functions. Furthermore, we demonstrated that the aptamer inhibits blood coagulation by interfering with the extrinsic pathway, blocking the complex of factor VIIa and tissue factor interactions with factor IX. The results from the previous and present studies suggest that the aptamer probably binds in the vicinity of the EGF1 and EGF2 domains of factor IX.

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A potent anticoagulant protein, IX-bp (Factor IX binding protein), has been isolated from the venom of Trimeresurus flavoviridis (habu snake) and is known to bind specifically to the Gla (gamma-carboxyglutamic acid-rich) domain of Factor IX. To evaluate the molecular basis for its anticoagulation activity, we assessed its interactions with various clotting factors. We found that the anticoagulation activity is primarily due to binding to the Gla domains of Factors IX and X, thus preventing these factors from recognizing phosphatidylserine on the plasma membrane. The present study suggests that ligands that bind to the Gla domains of Factors IX and X may have the potential to become novel anticoagulants.

A membrane-associated prothrombin activator (MAPA) was found on various cultured cells derived from non-hematopoietic cells [Sekiya, F. et al. (1994) J. Biol. Chem. 269, 32441-32445]. In this study, we investigated the enzymatic properties of this enzyme using protease inhibitors. While the metalloproteinase inhibitor, o-phenanthroline, had no effect, some Kunitz type serine protease inhibitors attenuated MAPA activity. Recombinant tissue factor pathway inhibitor (rTFPI) also markedly reduced the activity (IC(50), 1. 3+/-0.6 x 10(-10) M). MAPA activity is, therefore, most likely to be due to factor Xa. We evaluated the effect of exogenous factor Xa on MAPA activity. Factor Xa-dependent prothrombin activation was observed on fibroblast cells (apparent K(d), 1.47+/-0.72 nM). Activation was also observed on glial and neuronal cells, which expressed MAPA activity. These results imply that membrane-bound factor Xa results in MAPA activity on these cells. Therefore, we considered the involvement of factor Va, a component of prothrombinase, in this activity. We examined whether or not the prothrombinase complex is assembled on these cells. Prothrombin was activated in a manner dependent on both exogenous factor Xa and factor Va (apparent K(d) of 0.51-1.81 nM for factor Va). These results indicate that the prothrombinase complex forms specifically on various extravascular cells. Although the prothrombinase complex can be assembled on monocytes and lymphocytes, it is not known why these cells can activate prothrombin specifically. These cells which have the capacity for prothrombin activator activity could also activate factor X; i.e. cells with factor X activation activity were able to convert prothrombin. These observations suggest that thrombin was generated via two procoagulant activities; factor X activation and subsequent prothrombinase complex formation on the surface of these cells. This mechanism may explain the various pathological states involving or resulting from extravascular thrombin and fibrin formation.