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Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa. The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI. We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region. Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e. the residues 104 and 197, participates in the interaction with FXa in the quaternary complex. Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.Keywords: fluorescence; local probing; protein-protein interactions; site-directed labeling.Complex formation between factor VIIa (FVIIa) and its cofactor tissue factor (TF) triggers blood coagulation. The TF-FVIIa complex activates factor X (FX) to factor Xa (FXa) and factor IX (FIX) to factor IXa (FIXa), which both contribute to the formation of thrombin and ultimately a fibrin clot. Tissue factor pathway inhibitor (TFPI) is the prominent physiological inhibitor of the TF-FVIIa complex [1]. It is a plasma proteinase inhibitor composed of three Kunitz-type domains [2]. TFPI inhibits TF-FVIIa in two steps: first, binding occurs between the second Kunitz domain of TFPI and the active site of FXa; thereafter, the first Kunitz domain of TFPI binds to TF-bound FVIIa, and FXa-TFPI thereby causes feedback inhibition of TF-FVIIa. FXa-mediated TFPI-induced inhibition of TF-FVIIa catalytic activity results in formation of the stable quaternary complex TF-FVIIa-FXa-TFPI [3]. In addition to down-regulating the procoagulant function of TF-FVIIa, the TF-FVIIa-FXa-TFPI complex is also crucial for the cell-surface redistribution of inhibited TF-FVIIa complexes into caveolae [4], and the rate of internalization of TF-FVIIa is increased in some cell types when part of the quaternary complex with FXa-TFPI [5]. The role of the third Kunitz domain of TFPI is not yet fully understood, although it is known that TFPI(1-161) lacking this region can inhibit TF-FVIIa in complex with FXa [6].According to the X-ray crystallographic structure, FVIIa binds to the extracellular domain of TF in an extended conformation comprising contacts all the way from the lower part of the protease domain of FVIIa and the N-terminal domain of TF to the c-carboxyglutamic acid (Gla)-rich FVIIa module and the C-termin...

A versatile total synthesis of sialylgalactosylgloboside (SGG, 1), carrying the stage-specific embryonic antigen 4 (SSEA-4) is reported, illustrating a more general strategy for the synthesis of complex globo-series glycosphingolipids. Starting from readily available building blocks 7, 8, and 10, two different approaches to the synthesis of the key tetrasaccharide 6 have been developed in a highly convergent manner. Further glycosylations with galactosyl trichloroacetimidate (5) and sialyl phosphite (2) donors successively afforded the penta- and hexasaccharides 3 and 11. The latter was finally converted into the target molecule (SGG, 1) with the help of a azidosphingosine glycosylation procedure, favored in this case by the stereocontrolling properties of the 2a-O-pivaloyl protecting group. Valuable intermediates 6 and 3, having the oligosaccharidic skeletons of Gb(4) and Gb(5) (SSEA-3), respectively, were obtained in the course of the synthesis.

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Bioimpedance spectroscopy can identify pathological changes related to precancerous lesions of the cervix uteri and esophagus. It therefore has the potential to detect early reflux-related changes in the esophageal mucosa, such as dilated intercellular spaces. The reliable detection of dilated intercellular spaces at the time of endoscopy would yield a significant diagnostic advantage for separating patients with functional heartburn from the large proportion of patients with gastroesophageal reflux symptoms but no macroscopic esophagitis or pathological acid exposure. The bioimpedance of the esophageal mucosa, measured with a small caliber probe, was evaluated in a series of preclinical experiments. First, sections of rabbit esophageal epithelium were mounted in Ussing chambers and exposed to solutions at pH 7.4 or pH 1.5 for 45 minutes. Impedance measurements were taken at varying probe pressures. Second, rabbit esophageal epithelia were perfused for 45 minutes in situ with pH 1.1 or control solutions and impedance measurements taken. Samples from both in vitro and in situ experiments were taken for morphological examination by light microscopy. Finally, esophageal bioimpedance was measured in awake dogs with permanent esophagocutaneous stoma. The in situ experiments demonstrated that morphological changes in the esophageal mucosa could be discerned by the use of bioimpedance spectroscopy. The variability in resistivity was species-independent but was affected by the pressure applied to the probe. The results suggest that evaluation of bioimpedance spectroscopy for use in a clinical setting is warranted. Small morphological differences in the esophageal mucosa may be detected by the use of bioimpedance spectroscopy.