Blood cell progenitors were scanned for the presence of the coagulation starter protein tissue factor (TF) by immunoelectron microscopy. Thereby, substantial TF expression was observed in the precursor cells of eosinophils. TF levels were lower in basophil precursors and barely detectable in neutrophil progenitors. In peripheral blood immediately processed to avoid activation of the TF gene, mature eosinophils were found to considerably express TF, unique among the granulocyte and monocyte fractions. TF was preferentially located in the specific granules in resting eosinophils. Platelet-activating factor (PAF), and more pronounced, granulocyte-macrophage colony-stimulating factor (GM-CSF) plus PAF, caused translocation of preformed TF to the eosinophil cell membrane. GM-CSF/PAF also increased the TF transcript levels. The activated eosinophils exhibited procoagulant activity that was abrogated by TF inhibition. Targeting the extracellular domain of TF with specific antibodies markedly suppressed the initial phase of the eosinophil passage across the IL-4-activated endothelium. Eosinophil rolling and firm adhesion remained unaffected. This suggests that TF specifically facilitates the early transendothelial migration of the eosinophils. In summary, eosinophils maintain a high TF expression during maturation, providing a main source of preformed TF in blood, which might be relevant for the thrombogenesis promoted by hypereosinophilic conditions. IntroductionTissue factor (TF), the crucial starter protein of hemostasis and a major determinant of its pathologic sequelae, 1 is basically expressed in the plasma membrane of cells located in the adventitial and medial layers of the vascular wall. TF binds the serine protease factor VIIa with high affinity, thereby enhancing its proteolytic activity by several orders of magnitude, whereby coagulation is initiated. A series of new findings suggests that preformed TF is present in intravascular compartments in humans, at variance with its designation. For a deeper understanding of the start process of intravascular coagulation, it is essential to know where TF is located and how its expression is regulated. Recently, circulating microvesicles (or microparticles) were revealed as source for preexisting TF in blood, which are apparently derived from leukocytes and platelets. 2-5 They most likely represent the earlier described TF pool in acellular plasma 6-9 because the selective removal of microvesicles strongly decreases the plasma TF contents. 3 Microvesicles are rapidly recruited to the site of vascular injury in vivo, where they elicit the coagulation start in a TF-dependent way. 10 Moreover, TF has been reported to appear on the cell surface and on microvesicles secreted from activated platelets. [11][12][13] In other work, TF was not detectable on platelets. 14 In addition, TF has been proposed to be present in neutrophils (summarized by Nakamura et al 15 ), although this has been called into question. 16 Notably, the functional competence of TF is not restricted to ...

Blood coagulation in vivo is initiated by factor VII (FVII) binding to its cellular receptor tissue factor (TF). FVII is the only known ligand for TF, so it was expected that FVII-deficient embryos would have a similar phenotype to TF-deficient embryos, which have defective vitello-embryonic circulation and die around 9.5 days of gestation. Surprisingly, we find that FVII-deficient (FVII-/-) embryos developed normally. FVII-/- mice succumbed perinatally because of fatal haemorrhaging from normal blood vessels. At embryonic day 9.5, maternal-fetal transfer of FVII was undetectable and survival of embryos did not depend on TF-FVII-initiated fibrin formation. Thus, the TF-/- embryonic lethal and the FVII-/- survival-phenotypes suggest a role for TF during embryogenesis beyond fibrin formation.

Tissue factor (TF), a 47 kDa transmembrane glycoprotein, is the essential receptor and cofactor for factor VII/VIIa. Its distribution in normal tissues and in tumours has been recently investigated immunohistochemically with monoclonal and polyclonal anti-TF antibodies in frozen sections. The cardinal problem of this technique is the difficulty of determining exactly the localization of the reaction product at least in certain tissues. Here, we demonstrate a method using monoclonal anti-TF antibodies to detect TF in routinely fixed, microwaved, paraffin-embedded tissues. Generally, there were no fundamental differences in TF distribution in frozen and paraffin-embedded material. However, in most cases, the paraffin sections allow a better cellular localization of TF. For example, the staining pattern for TF in both kinds of sections is identical in kidney, brain and skin. The paraffin-embedded material, however, clearly shows that TF expression is restricted to the parietal and the visceral epithelia of Bowman's capsule of glomeruli in the kidney, and to astrocytes and their processes in the brain. TF reactivity in the skin is revealed to be cell membrane-bound; in cardiomyocytes TF shows an exclusively sarcolemmal localization. The immunohistological detection of TF in paraffin sections is a powerful tool for systematic studies on the possible role of TF in the context of physiological and pathological studies.

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An in vitro model consisting of endothelium grown on collagen was used to investigate how mononuclear phagocytes traverse endothelium in the basal-to-apical direction (reverse transmigration), a process that mimics their migration across vascular and/or lymphatic endothelium during atherosclerosis and resolution of inflammation, respectively. Monoclonal antibody (MoAb) VIC7 against tissue factor (TF) inhibited reverse transmigration by 77%. Recombinant tissue factor fragments containing at least six amino acids C-terminal to residue 202 also strongly inhibited reverse transmigration. TF was absent on resting monocytes but was induced on these cells after initial apical-to-basal transendothelial migration. Two additional observations suggest that TF is involved in adhesion between mononuclear phagocytes and endothelium: (1) when monocytes were incubated with lipopolysaccharide (LPS) to stimulate expression of TF before they were added to endothelium, VIC7 or soluble TF modestly inhibited their adhesion to the apical endothelial surface, each by about 35%; and (2) endothelial cells specifically bound to surfaces coated with TF fragments containing amino acids 202-219. This binding was blocked by anti-TF MoAb, suggesting that endothelial cells bear a receptor for TF. These data suggest that mononuclear phagocytes use TF, perhaps as an adhesive protein, to exit sites of inflammation.